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At least 19 records

Evaluation of Minimum Detectable Activities for Stack Sample Analyses

The Rad-NESHAP program takes samples at major point sources to track how much of select nuclides are being emitted out of the stack. Samples are taken at the site where the radiological operations are being performed. The samples are then sent to analytical laboratories for analysis to determine what nuclides exist in the sample. However, due to the sensitivity of the instruments, false positives are often reported. To reduce the number of false positives, the results reported from the analytical labs are compared to set detection limits. Anything below the set detection limit is considered part of the background while anything above the limit is considered a positive hit. Therefore, the detection limit must be set low enough that any false negatives do not result in a significant dose missed at the end of the year but high enough that there are not enough false positives to skew the annual dose being reported. All detection limits listed in this section for the analyses below were designed to meet the stated equivalent emissions rate and subsequent off-site doses. These minimum detectable activities (MDAs) were developed in the 1990s and documented in the memo cited for each analysis below. These limits and memos are all currently referenced in the Quality Assurance Project Plan (QAPP) for the Rad-NESHAP Compliance Task, which is being updated to a Project Implementation Plan (PIP) concurrently with this document. As part of this update process, the original MDAs defined in this section needed to be verified that they were still conservative and meet the off-site dose levels stated.

2019 MDA↗

TRAF2 regulates the cytoplasmic/nuclear distribution of TRAF4 and its biological function in breast cancer cells

Highlights: •TRAF2 appears to interact with TRAF4 in breast cancer cell lines. •TRAF2 affects the localization and function of TRAF4 in breast cancer cell lines. •TRAF4 may play an important role in the activation of NF-κB via TRAF2. -- Abstract: Although numerous studies have shown that tumor necrosis factor receptor-associated factor 4 (TRAF4) plays an important role in the carcinogenesis of many tumor types, its exact molecular mechanism remains elusive. In this study, we examined the regulation function of TRAF2 to the cytoplasmic/nuclear distribution of TRAF4 in the breast cancer cell line. Using cell immunofluorescent staining, we found that TRAF2 and TRAF4 were co-localized to the cytoplasm in MCF-7 cells. Co-immunoprecipitation showed that TRAF2 could interact with TRAF4 in MCF-10A, MCF-7 and MDA-MB-231 cell lines. Western blotting showed TRAF2 depletion by targeted siRNA in MDA-MB-231 cells led to reduced TRAF4 expression in the cytoplasm and augmented TRAF4 expression in the nucleus. Cytoplasmic expression of TRAF4 was augmented and nuclear expression was reduced when MCF-7 cells were transfected with hTRAF2pLPCX-HA-Flag/P874. MCF-7 cells expressing hTRAF2pLPCX-HA-Flag/P874 had enhanced cell proliferation rates. The nuclear expression of NF-κB significantly increased after TNF-α treatment. When hTRAF2pLPCX-HA-Flag/P874 and the siRNA-TRAF4 plasmid were cotransfected, the nuclear expression of NF-κB was significantly reduced compared with cells transfected with hTRAF2pLPCX-HA-Flag/P874 only. In conclusion, TRAF2 appears to interact with TRAF4 and affect the localization of TRAF4 in breast cancer cell lines. The overexpression of TRAF2 augmented the cytoplasmic expression of TRAF4 which promoted cell proliferation and inhibited cell apoptosis by activating NF-κB nuclear transcription. TRAF4 may play an important role in the activation of NF-κB via TRAF2.

60 APPLIED LIFE SCIENCES↗

LincRNA-RoR/miR-145 promote invasion and metastasis in triple-negative breast cancer via targeting MUC1

Highlights: • LincRNA-ROR was upregulated in TNBC cell lines and tissue samples. • LincRNA-ROR was able to significantly promote the invasion and metastasis of TNBC cell lines MDA-MB-231. • LincRNA-ROR/miR-145 regulated invasion and metastasis of MDA-MB-231 via the target MUC1. Triple-negative breast cancer (TNBC) was associated with high rates of cancer recurrence and metastasis and currently no available molecularly target. Accumulating evidences have established the importance of lincRNA-ROR as a marker of cancers. In order to better understand the mechanism of lincRNA-ROR in TNBC, we provided a novel molecular target into the regulatory invasion and metastasis in present research. We found that lincRNA-ROR was upregulated in TNBC cell lines and tissue samples. The aberrant expression of lincRNA-ROR was shown to increase invasion and metastasis in MDA-MB-231 and loss of function by siRNA reverse these process. Furthermore, lincRNA-ROR functions as a competing endogenous RNAs (ceRNA) which sponges miR-145 and therefore upregulate the expression of Mucin1 (MUC1). The expression of MUC1 impacted E-cadherin membrane localization. Together, MUC1 was a potential molecular target may help explain the role of lincRNA-ROR/miR-145 for invasion and metastasis in TNBC cell lines.

60 APPLIED LIFE SCIENCES↗

WE-FG-BRA-11: Theranostic Platinum Nanoparticle for Radiation Sensitization in Breast Cancer Radiotherapy

Purpose: We have developed a novel receptor-targeted theranostic platinum nanoparticle (HER-PtNP) for enhanced radiation sensitization in HER2-positive breast cancer radiation treatment. This study aims to evaluate receptor-targeting specificity, and radiation sensitization of the nanoparticle. Methods: The platinum nanoparticle (PtNP) was synthesized with the diameter of 2nm, and capped with cysteine. The nanoparticle was tagged with a fluorescent dye (cy5) for the fluoresence detection, and conjuated with HER2/3 targeted protein (HerPBK10) for HER2-targeting specificity. We evaluated the theranostic features using in vitro breast cancer cell models: HER2-positive BT-474, and HER2-negative MDA-MB-231. The HER2-targeting specificity was evaluated using immunofluorescence and confocal microscopy. For each cell line, three sets of samples, including non-stained control, fluorescence stained PtNP-cy5 treated, and HER-PtNP treated, were imaged by confocal microscopy. Two breast cancer cell lineages were incubated with PtNP and HER-PtNP at 10 µg/mL, and then irradiated with X-rays for 2 Gy dose at 50 kVp. A colonogenic assay was used to determine cellular survival fractions by immediately reseeding 300 cells after irradiation in growth media and allowing colonies to grow for 2 weeks. Results: The results of confocal images show that no apparent nanoparticle cellular uptake was observed in the HER2-(MDA-MB-231) cells with 1% for PtNP-cy5 and 0.5% for HER-PtNP. Similarly no apparent PtNP-cy5 uptake (<1%) for BT474 cells was observed. However, there was significant HER-PtNP uptake (73%) for the HER2+(BT474) cells. The clonogenic assay showed that BT474 cells treated with HER-PtNP had significantly lower survival compared to those treated with PtNP (32% vs 81%, p=0.01). However, no significant radiosensitivity enhancement was observed for MDA-MB-231 cell treated with PtNP and HER-PtNP (89% vs 92%, p=0.78). Conclusion: Our studies suggest that the HER2-targeted platinum nanoparticle has excellent receptor targeting specificity and enhanced radiation sensitization compared to nanoparticle alone, suggesting potential for clinical applications in breast cancer radiotherapy.

60 APPLIED LIFE SCIENCES↗

Alginate–peptide amphiphile core–shell microparticles as a targeted drug delivery system

We describe in this work the synthesis of microparticles with a doxorubicin drug conjugated alginate core and a shell of peptide amphiphile nanofibres functionalized for targeting the folate receptor. The spherical geometry of the particle core allows high drug loading per surface area, whereas the nanoscale fibrous shell formed by self-assembly of peptide amphiphiles offers a high surface to volume ratio that is ideal for targeting. Furthermore, the synthesised microparticles have a 60-fold higher cytotoxicity against MDA-MB-231 breast cancer cells compared to non-targeting particles.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Knockdown of long non-coding RNA TP73-AS1 suppresses triple negative breast cancer cell vasculogenic mimicry by targeting miR-490-3p/TWIST1 axis

Triple negative breast cancer (TNBC) is among the most clinically aggressive subtypes of breast cancer. Despite the availability of new drugs, patients suffering TNBC bear disheartening prognosis. Vasculogenic mimicry (VM) is a malignant tumor specific non-endothelial vascular network, which provide oxygen and nutrients to tumor cells and facilitate tumor progression. Therefore targeting TNBC VM formation may contribute to tumor treatment. In this study, we found that long non-coding RNA TP73 antisense RNA 1 (TP73-AS1) was upregulated in VM positive TNBC tissues. Knockdown of TP73-AS1 suppressed TNBC cell line MDA-MB-231 cell VM formation in vitro. In addition, RNA immunoprecipitation assay and dual luciferase reporter assay showed that TP73-AS1 bound to miR-490-3p in a sequence-specific manner. miR-490-3p was downregulated in VM positive tissues and was involved in TP73-AS1-mediated MDA-MB-231 cell VM formation. Furthermore, TWIST1 was a target of miR-490-3p and participated in TP73-AS1/miR-490-3p-modulated MDA-MB-231 cell VM formation. Findings in this study may provide insight in TNBC management.

60 APPLIED LIFE SCIENCES↗

Glutamine deprivation sensitizes human breast cancer MDA-MB-231 cells to TRIAL-mediated apoptosis

Tumor cell metabolism is a promising target for various cancer treatments. Apart from aerobic glycolysis, cancer cell growth is dependent on glutamine (Gln) supply, leading to their survival and differentiation. Therefore, we examined whether treatment with TNF-related apoptosis-inducing ligand (TRAIL) sensitizes MDA-MB-231 cells to apoptosis under Gln deprivation condition (TRAIL/Gln deprivation). Gln deprivation decreased cell proliferation as expected, but did not induce remarkable cell death. TRAIL/Gln deprivation, however, significantly increased growth inhibition and morphological shrinkage of MDA-MB-231 cells compared to those induced by treatment with either Gln deprivation or TRAIL alone. Moreover, TRAIL/Gln deprivation upregulated the apoptotic sub-G{sub 1} phase accompanied with a remarkable decrease of pro-caspase-3, pro-caspase-9, and anti-apoptotic xIAP, and Bcl-2. Increased cleavage of PARP and pro-apoptotic Bid protein expression suggests that TRAIL/Gln deprivation triggers mitochondrion-mediated apoptosis in MDA-MB-231 cells. Additionally, TRAIL/Gln deprivation upregulated the expression of endoplasmic reticulum (ER) stress markers such as ATF4 and phosphorylated eIF2α, thereby enhancing the C/EBP homologous protein (CHOP) protein level. Transient knockdown of CHOP partically reversed TRAIL/Gln deprivation-mediated apoptosis. Accordingly, TRAIL/Gln deprivation enhanced the expression of death receptor 5 (DR5) and transient knockdown of DR5 completely restored TRAIL/Gln deprivation-mediated apoptosis. Taken together, our results suggest that Gln deprivation conditions can be used for the development of new therapies for TRAIL-resistant cancers.

60 APPLIED LIFE SCIENCES↗

A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

60 APPLIED LIFE SCIENCES↗

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.

60 APPLIED LIFE SCIENCES↗

ATM inhibition induces synthetic lethality and enhances sensitivity of PTEN-deficient breast cancer cells to cisplatin

Highlights: • ATM is a synthetically lethal target in breast cancer cells with PTEN defects. • ATM inhibitor KU-60019 preferentially sensitizes PTEN-deficient breast cancer cells to cisplatin. • The selective cytotoxicity results from the increase of DNA damage accumulation and the decreased capability of DNA repair. • PTEN-deficient MDA-MB-468 cells have lower level of Rad51, higher ATM kinase activity than PTEN wild-type MDA-MB-231 cells. PTEN deficiency often causes defects in DNA damage repair. Currently, effective therapies for breast cancer are lacking. ATM is an attractive target for cancer treatment. Previous studies suggested a synthetic lethality between PTEN and PARP. However, the synthetically lethal interaction between PTEN and ATM in breast cancer has not been reported. Moreover, the mechanism remains elusive. Here, using KU-60019, an ATM kinase inhibitor, we investigated ATM inhibition as a synthetically lethal strategy to target breast cancer cells with PTEN defects. We found that KU-60019 preferentially sensitizes PTEN-deficient MDA-MB-468 breast cancer cells to cisplatin, though it also slightly enhances sensitivity of PTEN wild-type breast cancer cells. The increased cytotoxic sensitivity is associated with apoptosis, as evidenced by flow cytometry and PARP cleavage. Additionally, the increase of DNA damage accumulation due to the decreased capability of DNA repair, as indicated by γ-H2AX and Rad51 foci, also contributed to this selective cytotoxicity. Mechanistically, compared with PTEN wild-type MDA-MB-231 cells, PTEN-deficient MDA-MB-468 cells have lower level of Rad51, higher ATM kinase activity, and display the elevated level of DNA damage. Moreover, these differences could be further enlarged by cisplatin. Our findings suggest that ATM is a promising target for PTEN-defective breast cancer.

60 APPLIED LIFE SCIENCES↗

β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress. - Highlights: • Integrin β3 signaling promotes chemoresistance to epirubicin in breast cancer cells. • Integrin β3 promotes cell survival and proliferation in drug treated cells through the PI3K and MAPK pathways. • Integrin signaling helps evade drug induced cytotoxicity by repression of pro-apoptotic molecule; BAD.

60 APPLIED LIFE SCIENCES↗

Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

Highlights: Black-Right-Pointing-Pointer Matrigel alters cancer cell line miRNA expression relative to culture on plastic. Black-Right-Pointing-Pointer Many identified Matrigel-regulated miRNAs are implicated in cancer. Black-Right-Pointing-Pointer miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. Black-Right-Pointing-Pointer miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence of Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a valuable approach to the in vitro study of miRNAs.

60 APPLIED LIFE SCIENCES↗

Genome-wide profiles of metastasis-associated mRNAs and microRNAs in salivary adenoid cystic carcinoma

Highlights: • Different expressed miRNAs and mRNAs were profiled in lowly and highly metastatic SACC cell lines. • Multiple regulatory interactions between miRNAs and mRNAs may contribute to SACC metastasis. • miR-338–5p/3p and their target LAMC2 function on metastasis of multiple types of cancer. Salivary adenoid cystic carcinoma (SACC) is often accompanied with poor prognosis due to local recurrence, distant metastasis, and perineural invasion. The mechanism involved in SACC metastasis is not yet fully understood. In this study, we profiled the expression of messenger RNA (mRNA) and microRNA (miRNA) in a SACC cell line, ACC-2, and a highly metastatic SACC cell line, ACC-M, using high-throughput sequencing. We discovered that: (1) differentially expressed (DE) mRNAs and DE miRNAs are potentially involved in SACC metastasis; (2) multiple regulatory interactions between DE miRNAs and DE mRNAs exist; and (3) miR-338–5p/3p target LAMC2 to impair motility and invasion of ACC-M and MDA-MB-231 cells. In conclusion, our study integrated the regulatory effects of miRNAs and mRNAs on SACC metastasis and provided a potential application for miRNAs in future therapeutic intervention.

60 APPLIED LIFE SCIENCES↗

Sulphoraphane, a naturally occurring isothiocyanate induces apoptosis in breast cancer cells by targeting heat shock proteins

Highlights: Black-Right-Pointing-Pointer HSPs (27, 70 and 90) and HSF1 are overexpressed in MCF-7 and MDA-MB-231 cells. Black-Right-Pointing-Pointer Sulphoraphane, a natural isothiocyanate inhibited HSPs and HSF1 expressions. Black-Right-Pointing-Pointer Inhibition of HSPs and HSF1 lead to regulation of apoptotic proteins. Black-Right-Pointing-Pointer Alteration of apoptotic proteins activate of caspases particularly caspase 3 and 9 leading to induction of apoptosis. Black-Right-Pointing-Pointer Alteration of apoptotic proteins induce caspases leading to induction of apoptosis. -- Abstract: Heat shock proteins (HSPs) are involved in protein folding, aggregation, transport and/or stabilization by acting as a molecular chaperone, leading to inhibition of apoptosis by both caspase dependent and/or independent pathways. HSPs are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion and metastasis. HSPs particularly 27, 70, 90 and the transcription factor heat shock factor1 (HSF1) play key roles in the etiology of breast cancer and can be considered as potential therapeutic target. The present study was designed to investigate the role of sulphoraphane, a natural isothiocyanate on HSPs (27, 70, 90) and HSF1 in two different breast cancer cell lines MCF-7 and MDA-MB-231 cells expressing wild type and mutated p53 respectively, vis-a-vis in normal breast epithelial cell line MCF-12F. It was furthermore investigated whether modulation of HSPs and HSF1 could induce apoptosis in these cells by altering the expressions of p53, p21 and some apoptotic proteins like Bcl-2, Bax, Bid, Bad, Apaf-1 and AIF. Sulphoraphane was found to down-regulate the expressions of HSP70, 90 and HSF1, though the effect on HSP27 was not pronounced. Consequences of HSP inhibition was upregulation of p21 irrespective of p53 status. Bax, Bad, Apaf-1, AIF were upregulated followed by down-regulation of Bcl-2 and this effect was prominent in MCF-7 than in MDA-MB-231. However, very little change in the expression of Bid was observed. Alteration in Bcl-2 Bax ratio resulted in the release of cytochrome c from mitochondria and activation of caspases 3 and 9 which are in agreement with apoptotic index values. Sulphoraphane therefore can be regarded as a potent inducer of apoptosis due to HSP modulation in breast cancer cells.

60 APPLIED LIFE SCIENCES↗

GPER mediated estradiol reduces miR-148a to promote HLA-G expression in breast cancer

Highlights: • E2 induces the level of miR-148a in MCF-7 and MDA-MB-231 cells. • GPER mediates the E2-induced increase of miR-148a in MCF-7 and MDA-MB-231 cells. • E2-GPER regulates the expression of HLA-G by miR-148a. - Abstract: Breast cancer is the most common malignant diseases in women. miR-148a plays an important role in regulation of cancer cell proliferation and cancer invasion and down-regulation of miR-148a has been reported in both estrogen receptor (ER) positive and triple-negative (TN) breast cancer. However, the regulation mechanism of miR-148a is unclear. The role of estrogen signaling, a signaling pathway is important in development and progression of breast cancer. Therefore, we speculated that E2 may regulate miR-148a through G-protein-coupled estrogen receptor-1 (GPER). To test our hypothesis, we checked the effects of E2 on miR-148a expression in ER positive breast cancer cell MCF-7 and TN cancer cell MDA-MB-231. Then we used GPER inhibitor G15 to investigate whether GPER is involved in regulation of E2 on miR-148a. Furthermore, we analyzed whether E2 affects the expression of HLA-G, which is a miR-148a target gene through GPER. The results showed that E2 induces the level of miR-148a in MCF-7 and MDA-MB-231 cells, GPER mediates the E2-induced increase in miR-148a expression in MCF-7 and MDA-MB-231 cells and E2-GPER regulates the expression of HLA-G by miR-148a. In conclusion, our findings offer important new insights into the ability of estrogenic GPER signaling to trigger HLA-G expression through inhibiting miR-148a that supports immune evasion in breast cancer.

60 APPLIED LIFE SCIENCES↗

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+ ) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+ : mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗

Exosomes derived from breast cancer lung metastasis subpopulations promote tumor self-seeding

Highlights: • Tumor self-seeding is common in breast cancer. • Exosomes derived from MDA231-LM2 cells potentiate the growth of recipient tumors. • Exosomes derived from MDA231-LM2 cells favor the self-seeding of donor tumor cells. Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most patients with lung metastasis eventually die of recurrence. Recurrence may be related to self-seeding, which occurs when circulating tumor cells re-seed into the tumors they originated from (metastasis or carcinoma in situ). Tumor-derived exosomes have been intensively revealed to promote the progression of various cancers. However, whether tumor-derived exosomes play roles in tumor self-seeding has not yet been identified. By establishing a self-seeding nude mouse model, we found that exosomes derived from MDA231-LM2 cells (subpopulations of breast cancer lung metastasis) potentiate the growth of MDA-MB-231 xenografts. More importantly, laser confocal microscopy and flow cytometry results identified that MDA231-LM2-secreted exosomes promote the seeding of MDA231-LM2 cells into MDA-MB-231 xenografts. These findings suggest MDA231-LM2-secreted exosomes as a promising target to treat breast cancer lung metastasis.

60 APPLIED LIFE SCIENCES↗

EHMT2 is a metastasis regulator in breast cancer

Highlights: • EHMT2 (a histone methyltransferase) was determined to be significantly overexpressed in breast cancer tissues. • Knockdown of EHMT2 reduced cell migration/invasion and regulated the expression of EMT-related markers. • EHMT2 regulated EMT markers via up-regulation of MSK1-SNAIL1. Various modes of epigenetic regulation of breast cancer proliferation and metastasis have been investigated, but epigenetic mechanisms involved in breast cancer metastasis remain elusive. Thus, in this study, EHMT2 (a histone methyltransferase) was determined to be significantly overexpressed in breast cancer tissues and in Oncomine data. In addition, knockdown of EHMT2 reduced cell migration/invasion and regulated the expression of EMT-related markers (E-cadherin, Claudin 1, and Vimentin). Furthermore, treatment with BIX-01294, a specific inhibitor of EHMT2, affected migration/invasion in MDA-MB-231 cells. Therefore, our findings demonstrate functions of EHMT2 in breast cancer metastasis and suggest that targeting EHMT2 may be an effective therapeutic strategy for preventing breast cancer metastasis.

60 APPLIED LIFE SCIENCES↗