IL-7 splicing variant IL-7δ5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7δ5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7δ5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27(kip1) expression. Mechanistically, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7δ5. In conclusion, our findings demonstrate that IL-7δ5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target for human breast cancer therapeutics intervention.     

Synthesis and biological evaluation of histone deacetylase and DNA topoisomerase II-Targeted inhibitors

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities toward histone deacetylases and Topo II, and their cytotoxicities in cancer cell lines, were evaluated. Among the synthesized hybrid compounds, compound 16b showed the potent HDAC inhibitory activity at a low nanomolar level and exhibited antiproliferative activity toward cancer cell lines including MCF-7 (breast), HCT-116 (colon), and DU-145 (prostate) cancer cells at a low micromolar level. Moreover, compound 16a showed HDAC6-selectivity 20-fold over HDAC1.     

Synthesis, enzymatic inhibition, and cancer cell growth inhibition of novel delta-lactam-based histone deacetylase (HDAC) inhibitors

delta-Lactam-based hydroxamic acids, inhibitors of histone deacetylase (HDAC), have been synthesized via ring closure metathesis of key diene intermediates followed by conversion to hydroxamic acid analogues. The hydroxamic acids 12a, 12b, and 17c showed potent inhibitory activity in HDAC enzyme assay. The hydroxamic acid 12b exhibited growth inhibitory activity on five human tumor cell lines, showing good sensitivity on the MDA-MB-231 breast tumor cell.     

Chemotherapy cytotoxicity of human MCF-7 and MDA-MB 231 breast cancer cells is altered by osteoblast-derived growth factors

One-third of women with breast cancer will develop bone metastases and eventually die from disease progression at these sites. Therefore, we analyzed the ability of human MG-63 osteoblast-like cells (MG-63 cells), MG-63 conditioned media (MG-63 CM), insulin-like growth factor I (IGF-I), and transforming growth factor beta 1 (TGF-beta1) to alter the effects of adriamycin on cell cycle and apoptosis of estrogen receptor negative (ER-) MDA-MB-231 and positive (ER+) MCF-7 breast cancer cells, using cell count, trypan blue exclusion, flow cytometry, detection of DNA fragmentation by simple agarose gel, and the terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method for apoptosis (TUNEL assay). Adriamycin arrested MCF-7 and MDA-MB-231 cells at G2/M phase in the cell cycle and inhibited cell growth. In addition, adriamycin arrested the MCF-7 cells at G1/G0 phase and induced apoptosis of MDA-MB-231 cells. Exogenous IGF-I partially neutralized the adriamycin cytotoxicity/cytostasis of cancer cells. MG-63 CM and TGF-beta1 partially neutralized the adriamycin cytotoxicity of MDA-MB-231 cells but enhanced adriamycin blockade of MCF-7 cells at G1/G0 phase. MG-63 osteoblast-like cells inhibited growth of MCF-7 cells while promoting growth and rescued MDA-MB-231 cells from adriamycin apoptosis in a collagen co-culture system. These data suggest that osteoblast-derived growth factors can alter the chemotherapy response of breast cancer cells. Conceivably, host tissue (bone)-tumor cell interactions can modify the clinical response to chemotherapy in patients with advanced breast cancer.     

Salinomycin induces apoptosis and senescence in breast cancer: Upregulation of p21, downregulation of survivin and histone H3 and H4 hyperacetylation

Background

In the present study, we investigated the effect of Salinomycin on the survival of three human breast cancer cell lines MCF-7, T47D and MDA-MB-231 grown in adherent culture conditions.

Methods

Cell viability was measured by CellTiter-Glo and Trypan blue exclusion assay. Apoptosis was determined by caspase 3/7 activation, PARP cleavage and Annexin V staining. Cell cycle distribution was assessed by propidium iodide flow cytometry. Senescence was confirmed by measuring the senescence-associated β-galactosidase activity. Changes in protein expression and histone hyperacetylation was determined by western blot and confirmed by immunofluorescence assay.

Results

Salinomycinwas able to inhibit the growth of the three cell lines in time- and concentration-dependent manners. We showed that depending on the concentrations used, Salinomycin elicits different effects on theMDA-MB-231 cells. High concentrations of Salinomycin induced a G2 arrest, downregulation of survivin and triggered apoptosis. Interestingly, treatment with low concentrations of Salinomycin induced a transient G1 arrest at earlier time point and G2 arrest at later point and senescence associatedwith enlarged cellmorphology, upregulation of p21 protein, increase in histone H3 and H4 hyperacetylation and expression of SA-β-Gal activity. Furthermore, we found that Salinomycin was able to potentiate the killing of the MCF-7 and MDA-MB-231 cells, by the chemotherapeutic agents, 4-Hydroxytamoxifen and frondoside A, respectively.

Conclusion

Our data are the first to link senescence and histone modifications to Salinomycin.

Significance

This study provides a new insight to better understand the mechanism of action of Salinomycin, at least in breast cancer cells.     

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5–6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die. J. Cell. Physiol. 176:456-464, 1998. © 1998 Wiley-Liss, Inc.     

A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.     

TWIST represses estrogen receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.     

Effects of triptolide from Tripterygium wilfordii on ERα and p53 expression in two human breast cancer cell lines

The aim of the study was to discover possible differential cytotoxicity of triptolide towards estrogen-sensitive MCF-7 versus estrogen-insensitive MDA-MB-231 human breast cancer cells. Considering that MCF-7 cells express functional Estrogen receptor α (ERα) and wild-type p53, whereas MDA-MB-231 cells which are ERα-negative express mutant p53, the anti-proliferation effect of triptolide on MCF-7 and MDA-MB-231 cells were examined, the apoptotic effect and cell cycle arrest caused by triptolide were investigated, ERα and p53 expression were also observed in this paper. The results showed that the anti-proliferation effects were induced by triptolide in both cell lines. But the value of IC₅₀ in MCF-7 cells for its anti-proliferation effect was about one tenth of that in MDA-MB-231 cells, which indicated that the effect is more potent in MCF-7 cells. Condensed chromatin or fragmented nuclei could be found in MCF-7 cells treated with only 40 nM triptolide but in MDA-MB-231 cells they couldn’t be observed until the concentration reached to 400 nM. Triptolide induced significant S cell cycle arrest along with the presence of sub-G0/G1 peak in MDA-MB-231 cells, whereas there was only slightly S cell cycle arrest on cell cycle distribution in MCF-7 cells. The role of p53 in two breast cancer cells was examined, the results showed that the mutant p53 in MDA-MB-231 cells was suppressed and the wild-type p53 in MCF-7 was increased. Moreover, triptolide could down regulate the expression of ERα in MCF-7 cells. The results showed that triptolide is much more sensitive to ERα-positive MCF-7 cells than to ERα-negative MDA-MB-231 cells, and the sensitivity is significantly associated with the ERα and p53 status.     

HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.