Effects of metformin on breast cancer cell proliferation,the AMPK pathway and the cell cycle

Aim

The aim of this study was to compare the effects and mechanisms of action of metformin on estrogen receptor (ER)-positive and ER-negative breast cancer cell lines.

Methods

The anti-proliferative effects of metformin, and of the direct activator of adenosine monophosphate-activated protein kinase (AMPK), A-769662, on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) breast cancer cell lines were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a yellow tetrazole) assays. Fluorescence-activated cell sorting was also used to examine the effect of metformin on the cell cycle. Finally, phosphorylation of the metformin target AMPK, and of its potential downstream targets including acetyl-CoA carboxylase (ACC), p53, p70-S6K and Raptor, was examined using immunoblotting.

Results

Metformin and A-769662 caused significant, concentration-dependent suppression of cell proliferation with G1 cell cycle arrest in both MCF-7 and MDA-MB-231 cells. The proliferation suppression effect was more profound in MCF-7 cells. A concentration-dependent phosphorylation of AMPK was detected following metformin treatment, as was phosphorylation of ACC in both cell lines, but not p53, p70-S6k or Raptor.

Conclusion

Metformin acts as a growth inhibitor in both ER-positive and ER-negative breast cancer cells in vitro, and arrests cells in G1 phase, particularly in the ER-positive MCF-7 cells. The effect is likely to be mediated by AMPK activation, in part by inhibition of fatty acid synthesis via ACC phosphorylation.     

Antitumor and anticancer stem cell activity of a poly ADP-ribose polymerase inhibitor olaparib in breast cancer cells

Background

Although the poly adenosine diphosphate (ADP)-ribose polymerase (PARP) inhibitor olaparib is known to have potent antitumor activity in BRCA-related breast cancer cells, a limited number of preclinical and clinical studies have shown antitumor activity of olaparib in non-BRCA-related breast cancer. We investigated antitumor activity of olaparib in breast cancer cell lines derived from patients with nonfamilial sporadic breast cancer.

Methods

Effects of olaparib alone or in combination with five different chemotherapeutic agents on cell growth, cell cycle progression, apoptosis, and proportion of cancer stem cells using the mammosphere assay and CD44/CD24/ESA cell surface marker assay were investigated in a panel of six sporadic breast cancer cell lines. Extracellular-signal-regulated kinase (ERK) phosphorylation was also investigated to elucidate action mechanisms of olaparib.

Results

Olaparib inhibited the growth of two estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell lines and two ER-negative and HER2-negative breast cancer cell lines (50 % growth inhibitory concentrations 1.3–3.0 μM) associated with G2/M accumulation and induction of apoptosis. In contrast, two HER2-positive cell lines were resistant to olaparib. Interestingly, olaparib significantly decreased the proportion of putative cancer stem cells in either sensitive or resistant cell lines. In addition, olaparib increased expression of p-ERK. Combined treatments of olaparib with a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 completely suppressed expression of p-ERK. These treatments also inhibited the G2/M accumulation and apoptosis induction by olaparib. Among five chemotherapeutic agents commonly used for breast cancer treatment, only an irinotecan metabolite SN38 showed additive antitumor activity with olaparib. Importantly, the combined treatment enhanced the increase in G2/M accumulation and apoptosis induction as well as a decrease in the proportion of cancer stem cells.

Conclusions

This study has indicated for the first time that the PARP inhibitor olaparib has substantial antitumor and anticancer stem cell activity in breast cancer cell lines of nonfamilial origin. Upregulation of p-ERK might explain, at least in part, antitumor and anticancer stem cell activity of olaparib. Combined treatment of olaparib with irinotecan might be effective in treatment of non-BRCA-related breast cancer.     

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

Introduction

Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.

Methods

Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC₅₀ values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.

Results

Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D₁ were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G₀/G₁ arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.

Conclusions

These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991.     

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances taxol-induced cell death in breast cancer

Purpose

The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) enhances taxol-induced antitumor effects against some human cancer cells. The aim of this study is to investigate whether SAHA can enhance taxol-induced cell death against human breast cancer cells and to illustrate the mechanism in detail.

Methods

A panel of eight human breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the inhibitory effects of SAHA, taxol, or their combination by MTT assay. The effects of SAHA with or without taxol on cell cycle distributions, apoptosis, and protein expressions were also examined. The inhibitory effects on tumor growth were characterized in vivo in BALB/c nude mice bearing a breast cancer xenograft model.

Results

Taxol-resistant and multi-resistant breast cancer cells were as sensitive to SAHA as taxol-sensitive breast cancer cells. A dose-dependent synergistic growth inhibition was found in all the tested breast cancer cell lines treated with the SAHA/taxol combinations. The synergetic effect was also observed in the in vivo xenograft tumor model. The cell cycle analysis and apoptosis assay showed that the synergistic effects resulted from enhanced G2/M arrest and apoptosis.

Conclusions

SAHA increased the anti-tumor effects of taxol in breast cancer in vitro and in vivo. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.     

UBAP2L silencing inhibits cell proliferation and G2/M phase transition in breast cancer

Background

Ubiquitin-associated protein 2-like (UBAP2L) contains a ubiquitin-associated domain near its N-terminus, which has been demonstrated to be overexpressed in multiple tumors, including hepatocellular carcinoma and colorectal carcinoma but its role has not been well studied in breast cancer. Thus, this study was designed to evaluate whether UBAP2L can serve as a potential molecular target for breast cancer therapy.

Methods

The expression of UBAP2L was determined in breast cancer tissues and cell lines by Western blotting and Oncomine database mining. Then the expression of UBAP2L was silenced using RNA interference and the effects of UBAP2L knockdown on breast cancer cell proliferation and cell cycle progression by MTT and colony formation assay, and Flow cytometry, respectively.

Results

We found the expression of UBAP2L was significantly up-regulated in breast cancer tissues and cell lines. Knockdown of UBAP2L suppressed cell proliferation, impaired colony formation ability and induced cell cycle arrest at G2/M phase. At molecular levels, knockdown of UBAP2L increased p21 expression, but decreased the expression of CDK1 and Cyclin B1 in breast cancer cells.

Conclusion

Our findings suggest that UBAP2L plays an important role in breast cancer cell proliferation and might serve as a potential target for breast cancer treatment.

     

The investigation of Mitogen-Activated Protein kinase Phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging

Background

Breast cancer treatment is based on a combination of adjuvant chemotherapy followed by radiotherapy effecting intracellular signal transduction. With the tyrosine kinase inhibitors new targeted drugs are available. Imatinib mesylate is a selective inhibitor of bcr-abl, PRGFR alpha, beta and c-kit. The purpose of this study was to determine whether Imatinib has an influence on the effectiveness of radiotherapy in breast cancer cell lines and if a combination of imatinib with standard chemotherapy could lead to increased cytoreduction.

Methods

Colony-forming tests of MCF 7 and MDA MB 231 were used to study differences in cell proliferation under incubation with imatinib and radiation. Changes in expression and phosphorylation of target receptors were detected using western blot. Cell proliferation, migration and apoptosis assays were performed combining imatinib with doxorubicin.

Results

The combination of imatinib and radiotherapy showed a significantly stronger inhibition of cell proliferation compared to single radiotherapy. Differences in PDGFR expression could not be detected, but receptor phosphorylation was significantly inhibited when treated with imatinib. Combination of imatinib with standard chemotherapy lead to an additive effect on cell growth inhibition compared to single treatment.

Conclusions

Imatinib treatment combined with radiotherapy leads in breast cancer cell lines to a significant benefit which might be influenced through inhibition of PDGFR phosphorylation. Combining imatinib with chemotherapy enhances cytoreductive effects. Further in vivo studies are needed to evaluate the benefit of Imatinib in combination with radiotherapy and chemotherapy on the treatment of breast cancer.     

Anticancer effect of (E)-2-hydroxy-3��,4,5��-trimethoxystilbene on breast cancer cells by mitochondrial depolarization

Background

TMS (2,3??,4,5??-tetramethoxystilbene), a stilbene analog derived from rhapontigenin, was previously demonstrated to induce apoptosis in hormone-resistant breast cancer cells. Therefore, this study investigated the anticancer effect of a new stilbene analog, HTMS ((E)-2-hydroxy-3??,4,5??-trimethoxystilbene), and its mechanism in various breast cancer cell lines.

Materials and methods

The effect of HTMS on cell proliferation of MDA-MB-231, MCF-7, and LTED cells was evaluated using MTT assays. Cell apoptosis was detected by FITC-annexin V staining and flow cytometry analysis, changes in mitochondrial potential were determined by fluorescence microscopy using TMRE staining, and the expression of cleaved PARP and release of cytochrome c were assessed by Western blot analysis.

Results

HTMS significantly decreased the cell viability of various types of breast cancer cells in a dose- and time-dependent manner, characterized by G2/M arrest of the cell cycle and the induction of apoptosis. In particular, HTMS disturbed the mitochondrial membrane potential, causing a release of cytochrome c during apoptosis. Furthermore, HTMS was superior to TMS in inhibiting cancer cell growth in a pilot comparison study.

Conclusion

HTMS is an effective apoptotic agent for breast cancer cells, making it a candidate therapeutic agent for the treatment of breast cancer.     

Darbufelone, a novel anti-inflammatory drug, induces growth inhibition of lung cancer cells both in vitro and in vivo

Purpose

Inflammation plays a crucial role in the development of lung cancer. Accumulated studies have proved that non-steroidal anti-inflammatory drugs (NSAIDs) which block inflammation by their actions on arachidonic acid (AA) metabolism have a potential role in cancer chemotherapy and chemoprevention. The aim of our study was to investigate whether darbufelone, a novel anti-inflammatory drug, has anticancer effects in lung cancer.

Methods

Human non-small cell lung cancer cell lines were treated with darbufelone at various doses and time points for analysis of cell viability, cell cycle, and apoptosis in vitro. The in vivo effect of darbufelone was assessed in Lewis lung carcinoma mice model.

Results

Darbufelone inhibited the proliferation of non-small cell lung cancer cell lines in a dose-dependent manner, and induced cell cycle arrest at G0/G1 phase through up-regulation of p27 expression. Treatment with darbufelone also induced apoptosis by activating caspase-3 and caspase-8. Lewis lung carcinoma growth was also significantly inhibited by darbufelone treatment at daily dose of 80 mg/kg.

Conclusions

Taken together, these studies suggested that darbufelone, an anti-inflammation drug, might represent a novel therapeutic approach for lung cancer treatment.     

Stress hormones reduce the efficacy of paclitaxel in triple negative breast cancer through induction of DNA damage

Background:

The mechanisms by which stress hormones impact triple-negative breast cancer (TNBC) etiology and treatment are unclear. We have previously shown that stress hormones, cortisol, and catecholamines induce rapid DNA damage and impact DNA repair in NIH 3T3 fibroblasts. This study investigates whether stress hormones increase DNA damage in breast cancer cells and if this impacts drug efficacy.

Methods:

We first screened a panel of 39 breast cancer cell lines for expression of adrenergic and glucocorticoid receptors and examined if stress hormones induce DNA damage and alter cell cycle regulation in vitro. A TNBC xenograft model was used to assess the impact of restraint stress on tumour growth and chemosensitivity to paclitaxel.

Results:

We found that stress hormones induced DNA damage, phosphorylation of ATR, which was accompanied by an up-regulation of the G1 cell kinase inhibitor p21 and a cell cycle halt of TNBCs in the G1 phase. p21 knockdown abrogated G1 arrest by stress hormones. We also demonstrated that stress significantly decreased efficacy of paclitaxel.

Conclusion:

We describe a novel mechanism through which stress hormones can induce drug resistance to paclitaxel, which may have profound implications for treating drug resistance in patients with TNBC.     

In vitro sequence-dependent synergism between paclitaxel and gefitinib in human lung cancer cell lines

Purpose

In clinical trials, the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) administered concomitantly with first-line cytotoxicity chemotherapy failed to confer a survival benefit to patients with non-small-cell lung cancer (NSCLC). The aim of this study was to test whether paclitaxel followed by gefitinib is superior to other treatment schedules of NSCLC cell lines and to clarify the underlying mechanisms.

Methods

Human lung cancer cell lines with wild-type and mutant-type EGFR genes were used as in vitro models to define the differential effects of various schedules of paclitaxel with gefitinib treatment on cell growth, signaling pathway, and cell cycle distribution.

Results

Sequence-dependent antiproliferative effects differed between EGFR-TKI-resistant and EGFR-TKI-sensitive lung cancer cell lines. Exposure to paclitaxel resulted in an increased pEGFR level. This increase in phosphorylation was inhibited by subsequent exposure to gefitinib, whereas during the reverse sequence, the inhibition effect was reduced. After paclitaxel exposure, a higher level of pEGFR was observed in mitotic than in interphase cells. The sequence of paclitaxel followed by gefitinib resulted in greater anti-VEGF activity than did the reverse sequence. We confirmed that gefitinib arrested cells in G1, and paclitaxel arrested them in S phase. The sequence of paclitaxel followed by gefitinib arrested cells in G1, whereas the reverse sequence arrested cells in S and G2 phases.

Conclusions

These findings suggest that the sequence of paclitaxel followed by gefitinib may be superior to other sequences in treating NSCLC cell lines. Our results also provide molecular evidence to support clinical treatment strategies for patients with lung cancer.     

Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)

Introduction

Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified.

Methods

To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses.

Results

We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G₁-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level.

Conclusion

Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.     

Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

Introduction

The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

Methods

Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined.

Results

The percentage of CD44⁺/CD24^- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44⁺/CD24^-/low/ESA⁺. Furthermore, CD44⁺/CD24^-/ESA⁺ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy.

Conclusion

These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.     

Sensitivity of breast cancer cell lines to recombinant thiaminase I

Purpose

We have previously shown that the expression of the thiamine transporter THTR2 is decreased sevenfold in breast cancer, which may leave breast cancer cells vulnerable to acute thiamine starvation. This concept was supported by the observation that MDA231 breast cancer xenografts demonstrated growth inhibition in mice fed a thiamine-free diet.

Methods

We purified recombinant Bacillus thiaminolyticus thiaminase I enzyme, which digests thiamine, to study acute thiamine starvation in breast cancer.

Results

Thiaminase I enzyme was cytotoxic in six breast cancer cell lines with IC₅₀s ranging from 0.012 to 0.022 U/ml. The growth inhibitory effects of the combination of thiaminase I with either doxorubicin or paclitaxel were also examined. Over a wide range of drug concentrations, thiaminase 1 was consistently synergistic or additive with doxorubicin and paclitaxel in MCF-7, ZR75, HS578T and T47D cell lines, with most combinations having a calculated combination index (CI) of less than 0.8, indicating synergy. Although thiaminase I exposure did not stimulate the energy-sensing signaling kinases AKT, AMPK and GSK-3β in MCF-7, ZR75, HS578T and T47D cell lines, thiaminase I exposure did stimulate expression of the ER stress response protein GRP78. In summary, thiaminase I is cytotoxic in breast cancer cell lines and triggers the unfolded protein response.

Conclusion

These findings suggest that THTR2 down-regulation in breast tumors may present a nutritional vulnerability that could be exploited by thiaminase I enzyme therapy.     

Prolactin receptor antagonism reduces the clonogenic capacity of breast cancer cells and potentiates doxorubicin and paclitaxel cytotoxicity

Introduction

Exogenous prolactin is mitogenic and antiapoptotic in breast cancer cells, and overexpression of autocrine prolactin cDNA in breast cancer cell lines has been shown to stimulate their growth and to protect against chemotherapy-induced apoptosis. We examined the effects of the 'pure' prolactin receptor antagonist Δ1–9-G129R-hPrl (Δ1–9) on the breast cancer cell number and clonogenicity, alone and in combination with chemotherapy.

Methods

The effects of doxorubicin, paclitaxel and Δ1–9 on the growth of breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-468 and SK-BR-3) in monolayer culture were assessed by the sulphorhodamine B assay. Effects on clonogenicity were assessed by soft agar assay for the cell lines and by the mammosphere assay for disaggregated primary ductal carcinoma in situ samples. Dual-fluorescence immunocytochemistry was used to identify subpopulations of cells expressing the prolactin receptor and autocrine prolactin.

Results

Δ1–9 as a single agent had no effect on the cell number in monolayer culture, but potentiated the cytotoxic effects of doxorubicin and paclitaxel. Doxorubicin accordingly induced expression of prolactin mRNA and protein in all five breast cancer cell lines tested. Δ1–9 alone inhibited the clonogenicity in soft agar of cell lines by ~90% and the mammosphere forming efficiency of six disaggregated primary ductal carcinoma in situ samples by a median of 56% (range 32% to 88%). Subpopulations of cells could be identified in the cell lines based on the prolactin receptor and prolactin expression.

Conclusion

Autocrine prolactin appears to act as an inducible survival factor in a clonogenic subpopulation of breast cancer cells. The rational combination of cytotoxics and Δ1–9 may therefore improve outcomes in breast cancer therapy by targeting this cell population.     

Reversine induces cell cycle arrest,polyploidy, and apoptosis in human breast cancer cells

Background

Reversine, a small synthetic purine analogue, has been reported to be effective in tumor suppression. In the present study, we demonstrated an antitumor activity of reversine that could suppress cellular proliferation and induce cell cycle arrest and apoptosis in human breast cancer cell lines.

Methods

To evaluate whether reversine could suppress cell growth of MCF-7 and MDA-MB-231 cells and induce cell death, the cell viability, cell cycle, and apoptosis were determined in this study.

Results

Reversine treatment in human breast cancer cells reduced cell viability in a dose-dependent manner. Cell cycle accumulation at the G₂/M phase in reversine-treated cells was also determined. Moreover, polyploidy was also found in reversine-treated cells. Apoptosis in reversine-treated cells was exhibited with PARP cleavage and caspase-3 and caspase-8 activation, but not caspase-9 activation, indicating that caspase-dependent apoptosis mediated by an extrinsic pathway took place in reversine-treated cells. Furthermore, reversine attenuated cell death in cells pretreated with a pan-caspase inhibitor before reversine treatment.

Conclusions

In the present study, we demonstrated that reversine contributes to growth inhibition in human breast cancer cells through cell cycle arrest, polyploidy, and/or apoptosis induction. The apoptosis mediated by reversine was induced by the mitochondria-independent pathway. Therefore, the potential role of reversine as a novel therapeutic agent for the treatment of breast cancer is worthy of further investigation.     

Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo

Introduction

Adenine nucleotide translocator (ANT) 2 is highly expressed in proliferative cells, and ANT2 induction in cancer cells is known to be directly associated with glycolytic metabolisms and carcinogenesis. In addition, ANT2 repression results in the growth arrest of human cells, implying that ANT2 is a candidate for cancer therapy based on molecular targeting.

Methods

We utilized an ANT2-specific RNA interference approach to inhibit ANT2 expression for evaluating its antitumor effect in vitro and in vivo. Specifically, to investigate the therapeutic potential of ANT2 repression, we used a DNA vector-based RNA interference approach by expressing shRNA to knockdown ANT2 in breast cancer cell lines overexpressing ANT2.

Results

ANT2 shRNA treatment in breast cancer cell line MDA-MB-231 repressed cell growth as well as proliferation. In addition, cell cycle arrest, ATP depletion and apoptotic cell death characterized by the potential disruption of mitochondrial membrane were observed from the ANT2 shRNA-treated breast cancer cells. Apoptotic breast cancer cells transfected with ANT2 shRNA also induced a cytotoxic bystander effect that generates necrotic cell death to the neighboring cells. The intracellular levels of TNFα and TNF-receptor I were increased in ANT2 shRNA transfected cells and the bystander effect was partly blocked by anti-TNFα antibody. Ultimately, ANT2 shRNA effectively inhibited tumor growth in vivo.

Conclusion

These results suggest that vector-based ANT2 RNA interference could be an efficient molecular therapeutic method for breast cancer with high expression of ANT2.     

MiR-1301-3p inhibits human breast cancer cell proliferation by regulating cell cycle progression and apoptosis through directly targeting ICT1

Background

MiRNAs regulate a variety of biological processes, such as cell proliferation and apoptosis and play critical roles in cancer progression. Accumulating studies have demonstrated that miR-1301-3p could regulate the development and progression of multiple cancers, but its biological behaviors in breast cancer (BC) are still elusive.

Methods

The expression of miR-1301-3p was determined in BC tissues and cell lines using quantitative real-time PCR analysis. The effects of miR-1301-3p on BC cell growth, proliferation, cell cycle distribution, and apoptosis were also explored in vitro using MTT, colony formation and Flow cytometry assays. The potential target gene of miR-1301-3p was determined by dual-luciferase reporter assay and verified by quantitative real-time PCR and western blot analysis.

Results

We found the expression of miR-1301-3p was observably significantly down-regulated in BC tissues and cell lines. MiR-1301-3p expression in BC tissues was significantly associated with tumor size and clinical stage. Gain-of-function assays demonstrated that miR-1301-3p inhibited the cell growth and proliferation in breast cancer cell lines, MCF-7 and T-47D. Moreover, up-regulation of miR-1301-3p induced cell cycle G0/G1 phase arrest and apoptosis. Mechanistically, up-regulation of miR-1301-3p reduced the expression of CDK4, Cyclin D1, Bcl-2, but elevated the expression of p21, Bad and Bax. ICT1 was confirmed as a direct target of miR-1301-3p. Furthermore, ICT1 overexpression could partially reverse the effects of miR-1301-3p on BC cell proliferation, cell cycle progression and apoptosis.

Conclusion

Our observations suggested that miR-1301-3p inhibits cell proliferation via inducing cell cycle arrest and apoptosis through targeting ICT1, and might be a therapeutic target for BC.