Sensitivity Of Breast Cancer Cells Research Articles

OCT4B1 Dysregulation Is a Key Factor in Different Sensitivity of Normal Cells and Breast Cancer Cells to the Curcumin

OCT4B1 Dysregulation Is a Key Factor in Different Sensitivity of Normal Cells and Breast Cancer Cells to the Curcumin

MED27 plays a tumor-promoting role in breast cancer progression by targeting KLF4.

The mediator complex usually cooperates with transcription factors to be involved in RNA polymerase II-mediated gene transcription. As one component of this complex, MED27 has been reported in our previous studies to promote thyroid cancer and melanoma progression. However, the precise function of MED27 in breast cancer development remains poorly understood. Here, we found that MED27 was more highly expressed in breast cancer samples than in normal tissues, especially in triple-negative breast cancer, and its expression level was elevated with the increase in pathological stage. MED27 knockdown in triple-negative breast cancer cells inhibited cancer cell metastasis and stemness maintenance, which was accompanied by downregulation of the expression of EMT- and stem traits-associated proteins, and vice versa in non-triple-negative breast cancer. Furthermore, MED27 knockdown sensitized breast cancer cells to epirubicin treatment by inducing cellular apoptosis and reducing tumorsphere-forming ability. Based on RNA-seq, we identified KLF4 as the possible downstream target of MED27. KLF4 overexpression reversed the MED27 silencing-mediated arrest of cellular metastasis and stemness maintenance capacity in breast cancer in vitro and in vivo. Mechanistically, MED27 transcriptionally regulated KLF4 by binding to its promoter region at positions -156 to +177. Collectively, our study not only demonstrated the tumor-promoting role of MED27 in breast cancer progression by transcriptionally targeting KLF4, but also suggested the possibility of developing the MED27/KLF4 signaling axis as a potential therapeutic target in breast cancer.

LncRNA BREA2 promotes metastasis by disrupting the WWP2-mediated ubiquitination of Notch1

Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.

YBX1/lncRNA SBF2-AS1 interaction regulates proliferation and tamoxifen sensitivity via PI3K/AKT/MTOR signaling in breast cancer cells.

Y-box binding protein 1 (YBX1) is a multifunctional oncoprotein that can interact with several long non-coding RNAs (lncRNAs)to regulate metastasis in malignancies including breast cancer (BC). In the present study, we demonstrated the association of YBX1 with oncogenic lncRNA SBF2-AS1 (SET-binding factor 2 antisense RNA 1) via PI3K/AKT/mTOR signaling to regulate BC cell proliferation. We further explored the involvement of the YBX1/SBF2-AS1/PI3K/AKT/mTOR axis in the restoration of tamoxifen (TAM) sensitivity. YBX1-SBF2-AS1 association was predicted in silico and verified by RNA immunoprecipitation (RIP)-qPCR assay. Transfection experiments, Real-time RT PCR, Western blots, Phospho AKT/mTOR antibody array kit, and cell proliferation/apoptosis assays were employed to detect the YBX1/SBF2-AS1/ PI3K/AKT/mTOR axis and its effects upon TAM treatment in vitro. We identified that the YBX1 protein specifically binds to lncRNA SBF2-AS1. Our transfection experiments in MCF-7 and MDA-MB-468 cells with SBF2-AS1 silenced or overexpressed YBX1 plasmids, and their negative controls revealed that YBX1 regulates the expression of SBF2-AS1 by forming a positive feedback loop for its activation. We further demonstrated YBX1-SBF2-AS1 association exerts its effects on cell proliferation via PI3K/AKT/mTOR signaling pathway. Furthermore, we observed an increase in TAM sensitivity in BC cells after the knockdown of YBX1-SBF2-AS1 marked by decreased cell proliferation through disruption of the PI3K/AKT/mTOR axis. Our study has identified a novel YBX1/SBF2-AS1/PI3K/AKT/mTOR regulatory axis which may serve as a potential target to improve the effectiveness and efficacy of TAM treatment in BC.

Exosome-Transmitted tRF-16-K8J7K1B Promotes Tamoxifen Resistance by Reducing Drug-Induced Cell Apoptosis in Breast Cancer.

Tamoxifen resistance remains a challenge in hormone receptor-positive (HR+) breast cancer. Recent evidence suggests that transfer ribonucleic acid (tRNA)-derived fragments play pivotal roles in the occurrence and development of various tumors. However, the relationship between tRNA-derived fragments and tamoxifen resistance remains unclear. In this study, we found that the expression of tRF-16-K8J7K1B was upregulated in tamoxifen-resistant cells in comparison with tamoxifen-sensitive cells. Higher levels of tRF-16-K8J7K1B were associated with shorter disease-free survival in HR+ breast cancer. Overexpression of tRF-16-K8J7K1B promotes tamoxifen resistance. Moreover, extracellular tRF-16-K8J7K1B could be packaged into exosomes and could disseminate tamoxifen resistance to recipient cells. Mechanistically, exosomal tRF-16-K8J7K1B downregulates the expression of apoptosis-related proteins, such as caspase 3 and poly (ADP-ribose) polymerase, by targeting tumor necrosis factor-related apoptosis-inducing ligand in receptor cells, thereby reducing drug-induced cell apoptosis. Therapeutically, the inhibition of exosomal tRF-16-K8J7K1B increases the sensitivity of breast cancer cells to tamoxifen in vivo. These data demonstrate that exosomal tRF-16-K8J7K1B may be a novel therapeutic target to overcome tamoxifen resistance in HR+ breast cancer.

M6A reader HNRNPA2B1 destabilization of ATG4B regulates autophagic activity, proliferation and olaparib sensitivity in breast cancer

N6-methyladenosine RNA (m6A) is the most extensive epigenetic modification in mRNA and influences tumor progression. However, the role of m6A regulators and specific mechanisms in breast cancer still need further study. Here, we investigated the significance of the m6A reader HNRNPA2B1 and explored its influence on autophagy and drug sensitivity in breast cancer. HNRNPA2B1 was selected by bioinformatics analysis, and its high expression level was identified in breast cancer tissues and cell lines. HNRNPA2B1 was related to poor prognosis. Downregulation of HNRNPA2B1 reduced proliferation, enhanced autophagic flux, and partially reversed de novo resistance to olaparib in breast cancer. ATG4B was determined by RIP and MeRIP assays as a downstream gene of HNRNPA2B1, by which recognized the m6A site in the 3′UTR. Overexpression of ATG4B rescued the malignancy driven by HNRNPA2B1 in breast cancer cells and increased the olaparib sensitivity. Our study revealed that the m6A reader HNRNPA2B1 mediated proliferation and autophagy in breast cancer cell lines by facilitating ATG4B mRNA decay and targeting HNRNPA2B1/m6A/ATG4B might enhance the olaparib sensitivity of breast cancer cells.

ELOVL5 and IGFBP6 genes modulate sensitivity of breast cancer cells to ferroptosis.

Introduction: Relapse of breast cancer is one of the key obstacles to successful treatment. Previously we have shown that low expression of ELOVL5 and IGFBP6 genes in breast cancer tissue corresponded to poor prognosis. ELOVL5 participates directly in the elongation of polyunsaturated fatty acids (PUFAs) that are considered to play an important role in cancer cell metabolism. Thus, in this work we studied the changes in lipid metabolism in breast cancer cells with reduced expression of either ELOVL5 or IGFBP6 gene. Methods: MDA-MB-231 cells with a stable knockdown of either ELOVL5 or IGFBP6 gene were used in this study. Transcriptomic and proteomic analysis as well as RT-PCR were utilized to assess gene expression. Content of individual fatty acids in the cells was measured with HPLC-MS. HPLC was used for analysis of the kinetics of PUFAs uptake. Cell viability was measured with MTS assay. Flow cytometry was used to measure activation of apoptosis. Fluorescent microscopy was utilized to assess accumulation of ROS and formation of lipid droplets. Glutathione peroxidase activity was measured with a colorimetric assay. Results: We found that the knockdown of IGFBP6 gene led to significant changes in the profile of fatty acids in the cells and in the expression of many genes associated with lipid metabolism. As some PUFAs are known to inhibit proliferation and cause death of cancer cells, we also tested the response of the cells to single PUFAs and to combinations of docosahexaenoic acid (DHA, a n-3 PUFA) with standard chemotherapeutic drugs. Our data suggest that external PUFAs cause cell death by activation of ferroptosis, an iron-dependent mechanism of cell death with excessive lipid peroxidation. Moreover, both knockdowns increased cells' sensitivity to ferroptosis, probably due to a significant decrease in the activity of the antioxidant enzyme GPX4. Addition of DHA to commonly used chemotherapeutic drugs enhanced their effect significantly, especially for the cells with low expression of IGFBP6 gene. Discussion: The results of this study suggest that addition of PUFAs to the treatment regimen for the patients with low expression of IGFBP6 and ELOVL5 genes can be potentially beneficial and is worth testing in a clinically relevant setting.

Dynamic SUMOylation of MORC2 orchestrates chromatin remodelling and DNA repair in response to DNA damage and drives chemoresistance in breast cancer.

Rationale: SUMOylation regulates a plethora of biological processes, and its inhibitors are currently under investigation in clinical trials as anticancer agents. Thus, identifying new targets with site-specific SUMOylation and defining their biological functions will not only provide new mechanistic insights into the SUMOylation signaling but also open an avenue for developing new strategy for cancer therapy. MORC family CW-type zinc finger 2 (MORC2) is a newly identified chromatin-remodeling enzyme with an emerging role in the DNA damage response (DDR), but its regulatory mechanism remains enigmatic. Methods: In vivo and in vitro SUMOylation assays were used to determine the SUMOylation levels of MORC2. Overexpression and knockdown of SUMO-associated enzymes were used to detect their effects on MORC2 SUMOylation. The effect of dynamic MORC2 SUMOylation on the sensitivity of breast cancer cells to chemotherapeutic drugs was examined through in vitro and in vivo functional assays. Immunoprecipitation, GST pull-down, MNase, and chromatin segregation assays were used to explore the underlying mechanisms. Results: Here, we report that MORC2 is modified by small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 at lysine 767 (K767) in a SUMO-interacting motif dependent manner. MORC2 SUMOylation is induced by SUMO E3 ligase tripartite motif containing 28 (TRIM28) and reversed by deSUMOylase sentrin-specific protease 1 (SENP1). Intriguingly, SUMOylation of MORC2 is decreased at the early stage of DNA damage induced by chemotherapeutic drugs that attenuate the interaction of MORC2 with TRIM28. MORC2 deSUMOylation induces transient chromatin relaxation to enable efficient DNA repair. At the relatively late stage of DNA damage, MORC2 SUMOylation is restored, and SUMOylated MORC2 interacts with protein kinase CSK21 (casein kinase II subunit alpha), which in turn phosphorylates DNA-PKcs (DNA-dependent protein kinase catalytic subunit), thus promoting DNA repair. Notably, expression of a SUMOylation-deficient mutant MORC2 or administration of SUMO inhibitor enhances the sensitivity of breast cancer cells to DNA-damaging chemotherapeutic drugs. Conclusions: Collectively, these findings uncover a novel regulatory mechanism of MORC2 by SUMOylation and reveal the intricate dynamics of MORC2 SUMOylation important for proper DDR. We also propose a promising strategy to sensitize MORC2-driven breast tumors to chemotherapeutic drugs by inhibition of the SUMO pathway.

CircRNA CircABCB1 Diminishes the Sensitivity of Breast Cancer Cells to Docetaxel by Sponging MiR‑153-3p.

Resistance to docetaxel is a major problem to the success of docetaxel-based therapies for breast cancer. The present study was to identify the role of circABCB1 in altering the docetaxel resistance properties. Reverse transcription-quantitative PCR (qRT-PCR) was performed to quantify circABCB1 and miR-153-3p. The effects of circABCB1 on the viability, apoptosis and migration/invasion of docetaxel-resistant and -sensitive cells were investigated by cell function experiments, including Cell Counting Kit-8 and Transwell assays. Correlation between circABCB1 and the docetaxel-treated outcome was analyzed by multivariate Cox regression analysis, in addition to Kaplan-Meier analysis of time to treatment failure (TTF). The targeting relationship between circABCB1 and miR-153-3p was predicted and verified by dual-luciferase reporter assay and RNA immunoprecipitation. CircABCB1 was highly expressed in cancerous tissues, as well as the docetaxel-sensitive group and cells. The overexpression of circABCB1 contributed to cell viability, docetaxel-resistance and migration/invasion, but inhibited apoptosis. CircABCB1 can sponge miR-153-3p. CircABCB1 contributed to the docetaxel resistance of breast cancer, maybe via the miR-153-3p.

Identification of KCNK1 as a potential prognostic biomarker and therapeutic target of breast cancer.

Breast cancer is the most common malignant cancer and is the second most common cause of cancer-related deaths among females worldwide. Thus, it warrants the urgent development of new therapeutic targets and strategies. Potassium channels are aberrantly expressed in various tumors and are related to tumor progression. However, studies on potassium channels in breast cancer remain limited. First, The Cancer Genome Atlas (TCGA) and Gene Set Enrichment Analysis (GSEA) were used to screen the differentially expressed potassium channels in breast cancer. Several other databases were utilized for further data analysis and visualization, including Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Human Protein Atlas (HPA), GeneMANIA, Tumor Immune Estimation Resource 2 (TIMER2), Catalog of Somatic Mutations in Cancer (COSMIC), cBioPortal, and UCSC Xena tool. Besides, cell proliferation was detected by cell counting kit-8 (CCK8) and 5-Ethynyl-20-deoxyuridine (EdU), and cell migration was detected by wound healing and Transwell assays after knocking down KCNK1. Furthermore, the effect of KCNK1 knockdown on the sensitivity of breast cancer cells to paclitaxel was also evaluated. KCNK1 was overexpressed in breast cancer. Higher KCNK1 expression predicted an unfavorable prognosis. Moreover, the abnormal expression of KCNK1 was attributed to promoter hypomethylation of KCNK1 in breast cancer. Besides, cell proliferation and migration were significantly inhibited post-KCNK1 silencing, while KCNK1 knockdown significantly increased breast cancer cell sensitivity to paclitaxel. Taken together, our findings demonstrated that KCNK1 is a potential prognostic biomarker and therapeutic target of breast cancer. Thus, targeting KCNK1 might help synergize with paclitaxel function in breast cancer treatment.

Telomerase Inhibition by MST-312 Sensitizes Breast Cancer Cells to the Anti-cancer Properties of Plumbagin.

Breast cancer is the most common cause of malignancy and the second most common cause of death due to cancer in women. This heterogeneous disease is currently broadly classified as estrogen receptor (ER), progesterone receptor (PR) positive luminal tumors, human epidermal growth factor receptor 2 (HER2) amplified tumors and triple-negative breast cancers (TNBC). Phytochemicals are proven to be promising anti-cancer chemotherapeutics agents with minimal cytotoxic effects on normal cells. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a phytochemical derived from the roots of Plumbago zeylanica and it is known to possess anti-cancer properties similar to other compounds of naphthoquinones. In about 90% of cancer cells, the telomerase enzyme activity is revived to add telomeric repeats to evade apoptosis. In this study, a combinatorial approach of combining the anti-cancer compound plumbagin to induce genotoxicity and a potent telomerase inhibitor, MST-312 (synthetic derivative of tea catechins), was used to determine the combinational treatment-induced lethality in breast cancer cells such as MDA-MB-231 (TNBC) and MCF-7 (lumina) cells. MDA-MB-231 cells were responsive to combination treatment in both short-term (48 h) and long-term treatment (14 days) in a synergistic manner, whereas in MCF-7, the combination treatment was more effective in the long-term regimen. Furthermore, the cytotoxic effects of the plumbagin and MST-312 combination treatment were not recoverable after the short-term treatment. In conclusion, a combination treatment of MST-312 and plumbagin is proven to be more effective than a single plumbagin compound treatment in inducing DNA damage and telomere dysfunction leading to greater genome instability, cell cycle arrest and eventually cell death in cancer cells.

Sensitizing Breast Cancer Cells Using Standard Microwave Devices and Temperature Monitoring With Non-Invasive Thermochemical Thermometers

To review the effect of combined radiotherapy and hyperthermia in the treatment of recurrent breast cancer, two microwave antennas were compared through <em>in vitro</em> experimental tests, and the patient's skin temperature was monitored with calibrated thermo chemical thermometers. The main purpose of this research is to study the influence of shape of microwave antennas on the temperature transmission. The secondary goal is to select treatment procedures to ensure Good Clinical Practice (GCP) of hyperthermia using non-invasive thermometers.

Blockade of store-operated calcium entry sensitizes breast cancer cells to cisplatin therapy via modulating inflammatory response

Store-operated calcium entry (SOCE) is an important pathway for calcium signaling that regulates calcium influx across the plasma membrane upon the depletion of calcium stores in the endoplasmic reticulum. SOCE participates in regulating a number of physiological processes including cell proliferation and migration while SOCE dysregulation has been linked with pathophysiological conditions such as inflammation and cancer. The crosslink between cancer and inflammation has been well-established where abundant evidence demonstrate that inflammation plays a role in cancer pathophysiology and the response of cancer cells to chemotherapeutic agents including cisplatin. Indeed, the efficacy of cisplatin against cancer cells is reduced by inflammation. Interestingly, it was shown that SOCE enhances inflammatory signaling in immune cells. Therefore, the main objectives of this study are to examine the impact of SOCE inhibition on the cisplatin sensitivity of breast cancer cells and to explore its related mechanism in modulating the inflammatory response in breast cancer cells. Our findings showed that SOCE inhibitor (BTP2) enhanced cisplatin cytotoxicity against resistant breast cancer cells via inhibition of cell proliferation and migration as well as induction of apoptosis. We also found an upregulation in the gene expression of two major components of SOCE, STIM1 and ORAI1, in cisplatin-resistant breast cancer cells compared to cisplatin-sensitive breast cancer cells. In addition, cisplatin treatment increased the gene expression of STIM1 and ORAI1 in cisplatin-resistant breast cancer cells. Finally, this study also demonstrated that cisplatin therapy caused an increase in the gene expression of inflammatory mediators COX2, IL-8, and TNF-α as well as COX2 protein and upon SOCE inhibition using BTP2, the effect of cisplatin on the inflammatory mediators was reversed. Altogether, this study has proven the pivotal role of SOCE in cisplatin resistance of breast cancer cells and showed the importance of targeting this pathway in improving breast cancer therapy.

MicroRNA-484 / Akt axis in the regulation of breast cancer cells sensitivity to antitumor drugs

The development of acquired resistance of malignant tumors to specific drugs, such as target and hormonal drugs, is usually associated with a rearrangement of the intracellular signaling network and activation of unblocked growth pathways. Epigenetic regulators, in particular, non-coding miRNAs that control the level of expression of specific signaling proteins, are directly involved in the development and maintenance of such changes. We have previously shown that the development of resistance of breast cancer cells to mTOR (mammalian target of rapamycin) inhibitors and hormonal drugs is accompanied by constitutive activation of protein kinase Akt, the key anti-apoptotic protein.Aim. To study the role of microRNAs in the regulation of Akt expression and the formation of a resistant phenotype of breast cancer cells.We have shown that Akt activation in the tamoxifen- or rapamycin-resistant MCF-7 sublines is associated with a decrease in the level of miRNA-484, one of the Akt suppressors. Transfection of microRNA-484 into MCF-7 cells does not affect the activity of estrogen signaling, but leads to a marked decrease in Akt expression and is accompanied by an increase in cell sensitivity to tamoxifen and rapamycin. The obtained data demonstrate the involvement of the miRNA-484 / Akt axis in the breast cancer cells’ sensitization to target and hormonal drugs, which allows us to consider miRNA-484 as a potential candidate for drug development to cure resistant cancers.

Inhibition of branched-chain alpha-keto acid dehydrogenase kinase augments the sensitivity of ovarian and breast cancer cells to paclitaxel

ContextMany cancer patients who initially respond to chemotherapy eventually develop chemoresistance, and to address this, we previously conducted a RNAi screen to identify genes contributing to resistance. One of the hits from the screen was branched-chain α-keto acid dehydrogenase kinase (BCKDK). BCKDK controls the metabolism of branched-chain amino acids (BCAAs) through phosphorylation and inactivation of the branched-chain α-keto acid dehydrogenase complex (BCKDH), thereby inhibiting catabolism of BCAAs.MethodsWe measured the impact on paclitaxel sensitivity of inhibiting BCKDK in ovarian and breast cancer cell lines.ResultsInhibition of BCKDK using siRNA or two chemical inhibitors (BCKDKi) was synergistic with paclitaxel in both breast and ovarian cancer cells. BCKDKi reduced levels of BCAA and the addition of exogenous BCAA suppressed this synergy. BCKDKi inactivated the mTORC1-Aurora pathway, allowing cells to overcame M-phase arrest induced by paclitaxel. In some cases, cells almost completed cytokinesis, then reverted to a single cell, resulting in multinucleate cells.ConclusionBCKDK is an attractive target to augment the sensitivity of cancer cells to paclitaxel.

Anticancer and chemosensitizing activities of stilbenoids from three orchid species.

Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2μM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2μM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.

Baicalein sensitizes triple negative breast cancer MDA-MB-231 cells to doxorubicin via autophagy-mediated down-regulation of CDK1.

Triple negative breast cancer (TNBC) is a kind of refractory cancer with poor response to conventional chemotherapy. Recently, the combination of baicalein and doxorubicin was reported to exert a synergistic antitumor effect on breast cancer. However, the underlying mechanism how baicalein sensitizes breast cancer cells to doxorubicin remains to be elucidated. Here, it was found that 20μM baicalein increased the autophagy markers including the ratio of LC3B II/I, GFP-LC3 punctate aggregates and down-regulation of p62 expression, and up-regulated mitophagy marker PINK1 and Parkin in TNBC MDA-MB-231 cells as well. In contrast, doxorubicin decreased the levels of autophagy markers, and significantly up-regulated CDK1 in MDA-MB-231 cells. Pretreatment with baicalein markedly inhibited the doxorubicin-induced decrease in autophagy markers and up-regulation of CDK1, which was reversed by the autophagy inhibitor 3-Methyladenine. Moreover, baicalein alleviated the doxorubicin-induced expression and phosphorylation (at Ser616) of mitochondrial fission protein Drp1. Intriguingly, the autophagy inhibitor 3-Methyladenine also significantly weakened the effect of baicalein on doxorubicin-induced viability decrease and apoptosis in MDA-MB-231 cells. Taken together, our data indicate that baicalein improves the chemosensitivity of TNBC cells to doxorubicin through promoting the autophagy-mediated down-regulation of CDK1, also suggest a novel strategy for prevention of TNBC in the future.

Cancer Cells and Platelets Have a Synergistic Role on the Acceleration of Clot Formation and Fibrin Polymerization. a Thromboelastometric Study

Introduction The histological type and the aggressiveness of cancer cells as well as the acquisition of resistance to chemotherapy are significant predictors for the high risk of cancer associated thrombosis (CAT). It is well established that cancer cells, by expressing tissue factor (TF) trigger thrombin generation. Cancer cell derived extracellular vesicles (CaCa-dEV) bearing TF, in the microenvironment, further enhance thrombin generation. The impact of cancer cells on clot formation kinetics and the qualitative characteristics of the clot has not been evaluated yet. Aim The present study aimed to analyze the variables which influence the kinetics and the qualitative characteristics of the clot after triggering thrombin generation by cells from pancreatic cancer (BXPC3), breast cancer (MCF7 and MCF7R10) and multiple myeloma (RPMI-8226 and U266) - associated with different risk of CAT. The impact of the resistance to chemotherapy on the clot characteristics was also studied. Methods Cancer cell lines (BXPC3, MCF7, MCF7R10, RPMI-8226 and U266) and primary umbilical vein endothelial cells (HUVEC) for the control experiments were used according to standardized and validated experimental procedure. Doxorubicin resistant breast cancer cells (MCF7R10) were obtained by treating native breast cancer cells (MCF7) by increasing concentrations of Doxorubicin from 5 nM to 10 µM.. Thromboelastometry, using the ROTEM® delta device (from Warfen Paris, France), was used for the study of clot formation and firmness in whole blood (WB), platelet rich and platelet poor plasma (PRP and PPP respectively) in the presence of cancer cells or HUVEC (control). A suspension of 200 μL with 105 cells was placed in each sterilized cup. Following an incubation of 4 hour, culture media was removed and 330 μL of WB, PRP or PPP were added followed by addition of 20 µM of CaCl2 (0.2 M). Clotting time (CT), α-angle and maximum clot firmness (MCF) were analyzed. Results In PPP, pancreatic cancer cells BXPC3 as well as chemotherapy sensitive and resistant breast cancer cells (MCF7 and MCF7R10), significantly reduced the CT. MCF7R10 cells significantly reduced the CT as compared to MCF7. In contrast, myeloma cells (RPMI8226 and U266), did not significantly affect the CT. All studied cancer cell lines significantly increased the α-angle as compared to HUVEC, with the most potent effect from BXPC3 cells. The studied cancer cell lines did not significantly affect MCF. In PRP, BXPC3 cells significantly reduced the CT whereas MCF7 and MCF7R10 did not significantly change the CT, and myeloma cells significantly prolonged the CT as compared to HUVEC. BXPC3 cells significantly increased the α-angle as compared to the HUVEC, whereas the other studied cancer cell lines did not have any significant effect on this parameter. BXPC3, MCF7 and MCF7R10 cells did not significantly affect the MCF whereas the myeloma cells significantly decreased the MCF as compared to HUVEC. In WB, BXPC3 and MCF7 cells significantly decreased the CT whereas MCF7R10 did not show any significant effect and the myeloma cells significantly prolonged it. The α-angle significantly increased in the presence of BXPC3 cells whereas the other cell lines did not show any significant effect on this parameter. The presence of cancer cells resulted in significant decrease of the MCF as compared to the control (HUVEC), with the most potent effect from myeloma. In the control experiment (HUVEC), the comparison of the thromboelastometric profile of the three experimental systems (PPP, PRP and WB) showed that platelets (PRP) had a substantial impact on CT, α-angle and MCF whereas the erythrocytes (in WB) did not further modify these parameters. The same pattern of the role of platelets was observed in the presence of the studied cancer cell lines. Discussion The present study provides experimental evidence documenting that cancer cells impact clot formation kinetics without affecting clot firmness. The impact of cancer cells on clot formation kinetics is determined principally by the histological type. The pancreatic adenocarcinoma cells have a more pronounced effect on clot formation kinetics as compared to the breast cancer cells (sensitive or resistant to Doxorubicine). In contrast, multiple myeloma cells delay fibrin formation and polymerization and decrease the clot firmness. Platelets have a synergistic effect with cancer cells on the acceleration of the clot formation kinetics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of aspirin on the TNF-α-mediated cell survival and death pathways in breast cancer.

Aspirin is an anti-inflammatory drug commonly used as an analgesic and in cardiovascular disorders. However, many studies have highlighted its anti-cancer properties, especially in colorectal, lung, head and neck, and breast cancers. In this work, we tried to study the effect of aspirin on the TNF-α-mediated cell survival and death pathways in two cell lines representing two different subtypes of breast cancer. TNF-α-mediated stimulation of a cell can result in its proliferation via theNF-κB pathway or its death via either apoptosis or a programmed form of necrosis called necroptosis. The latter is believed to come into the picture only when apoptosis is inhibited. In this work, we studied the effect of aspirin on the TNF-α-mediated cell survival pathway and observed a decrease in expression of the NF-κB pathway regulators, itsnuclear translocation, and phosphorylation in a dose-dependent manner. The effect of aspirin on the TNF-α-mediated cell death showed significant cytotoxicity at the higher doses (5-20mM) of aspirin in both the breast cancer cell lines. The effect of aspirin on necroptosis was investigated after stimulating the cells with TNF-α and inhibiting apoptosis using Z-VAD-FMK. Though no significant effect was noted in breast cancer cell lines, the above protocol successfully induced necroptosis in L929, i.e.,a positive control cell line for necroptosis having an intact necroptosis machinery. Even when combined with the chemotherapeutic drugs, the above regime failed to induce any significant necroptosis in breast cancer cells but was found effective in L929. Overall, the findings show that while aspirin has the potential to inhibit the TNF-α-mediated cell survival pathway, it does not help sensitize breast cancer cells to necroptotic cell death induction.

Integrin α6β4 signals through DNA damage response pathway to sensitize breast cancer cells to cisplatin.

Integrin α6β4 is highly expressed in triple negative breast cancer (TNBC) and drives its most aggressive traits; however, its impact on chemotherapeutic efficacy remains untested. We found that integrin α6β4 signaling promoted sensitivity to cisplatin and carboplatin but not to other chemotherapies tested. Mechanistic investigations revealed that integrin α6β4 stimulated the activation of ATM, p53, and 53BP1, which required the integrin β4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin α6β4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we found that in response to cisplatin-induced DNA double strand break (DSB), integrin α6β4 suppressed the homologous recombination (HR) activity and enhanced non-homologous end joining (NHEJ) repair activity. Finally, we discovered that integrin α6β4 preferentially activated DNA-PK, facilitated DNA-PK-p53 and p53-53BP1 complex formation in response to cisplatin and required DNA-PK to enhance ATM, 53BP1 and p53 activation as well as cisplatin sensitivity. In summary, we discovered a novel function of integrin α6β4 in promoting cisplatin sensitivity in TNBC through DNA damage response pathway.