Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells

The endoplasmic reticulum protein 29 (ERp29) has a critical role in regulating protein folding, maturation and secretion. However, its role in carcinogenesis remains elusive. Recently, we reported that ERp29 is a novel tumor suppressor and regulates mesenchymal-epithelial transition in MDA-MB-231 breast cancer cells. Here, we investigated whether ERp29 plays a role in the response of breast cancer cells to chemotherapeutic agents. We found that expression of ERp29 increased the resistance to doxorubicin, but not cisplatin and paclitaxel, and decreased the doxorubicin-induced cell apoptosis in MDA-MB-231 cells, whereas knockdown of ERp29 in MCF-7 cells increased the doxorubicin cytotoxicity. A proteomics study identified up-regulation of Hsp27 and down-regulation of stathmin-1, galectin and prohibitin in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells. Further, we demonstrated that ERp29 up-regulated expression of Hsp27 by down-regulating eukaryotic translational initiation factor 2α (eIF2α). When Hsp27 was knocked down by siRNA in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells and parental MCF-7 cells, cell viability was significantly decreased and doxorubicin-induced cell apoptosis was enhanced. These results indicate that Hsp27 is involved in the ERp29-mediated resistance to doxorubicin. Therefore, targeting of Hsp27, with a combination of other chemotherapeutic agents, is a rational strategy in treating doxorubicin-resistant cancer cells.     

SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells

Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.     

IKK-β mediates chemoresistance by sequestering FOXO3; a critical factor for cell survival and death

Chemotherapeutic drugs proved only 50% successful in breast cancer because of cell type-dependent resistance mechanisms. FOXO3 is known to be involved in the regulation of several cell death-related genes; however, the extent of FOXO3 regulation in chemoresistance is still not fully understood. Here, we show that FOXO3 critically mediates cisplatin chemosensitivity of MCF-7 breast cancer cells which express higher levels of FOXO3 compared to resistant MDA-MB-231 cells. Administration of cisplatin induces apoptosis in MCF-7 cells in a FOXO3-dependent manner as indicated by RNA interference. On the other hand, IKK-β (IκB kinase) appears to inhibit FOXO3 action after cisplatin treatment and promotes chemoresistance in MDA-MB-231 cells. IKK-β directly interacts and sequesters FOXO3 in the cytosol preventing its nuclear localization. Moreover, cisplatin treatment induces autophagosome formation through LC-3 conversion while inhibiting the cleavage of caspase 9 and caspase 3 in MDA-MB-231 cells manipulated to overexpress FOXO3. In brief, our findings demonstrate that in addition to cellular level of active FOXO3, cisplatin chemoresistance is also regulated by IKK-β sequestration of FOXO3 in cytosol.     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

Radiosensitizing effect of conjugated linoleic acid on tumor cells MCF-7 and MDA-MB-231

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO-1, the CLA-9cis 11cis at 50 micro mol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.     

Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells

BackgroundBreast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.Hypothesis/purposeThe present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellata in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.Study designThe inhibitory effects of Saxi A on cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.MethodsMTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.ResultsOur results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shERα-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ERα remained viable.ConclusionSaxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ERα status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.     

Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family

Members of the Bcl-2 family of apoptosis-regulating proteins contain at least one of the four evolutionarily conserved domains, termed BH1, BH2, BH3, or BH4. Here, we report the identification, cloning, physical mapping, and expression pattern of BCL2L12, a novel gene that encodes a BCL2-like proline-rich protein. Proline-rich sites have been shown to interact with Src homology region 3 (SH3) domains of several tyrosine kinases, mediating their oncogenic potential. This new gene maps to chromosome 19q13.3 and is located between the IRF3 and the PRMT1/HRMT1L2 genes, close to the RRAS gene. BCL2L12 is composed of seven coding exons and six intervening introns, spanning a genomic area of 8.8 kb. All of the exon-intron splice sites conform to the consensus sequence for eukaryotic splice sites. The BCL2L12 protein is composed of 334 amino acids, with a calculated molecular mass of 36.8 kDa and an isoelectric point of 9.45. The BCL2L12 protein contains one BH2 homology domain, one proline-rich region similar to the TC21 protein and, five consensus PXXP tetrapeptide sequences. BCL2L12 is expressed mainly in breast, thymus, prostate, fetal liver, colon, placenta, pancreas, small intestine, spinal cord, kidney, and bone marrow and to a lesser extent in many other tissues. We also identified one splice variant of BCL2L12 that is primarily expressed in skeletal muscle.     

Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.     

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

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

GSK3β regulates Bcl2L12 and Bcl2L12A anti-apoptosis signaling in glioblastoma and is inhibited by LiCl

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b binding partner in a testis cDNA library. Our data demonstrated that GSK3b interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12_153–191 fragment located outside of the C-terminal BH2 motif is responsible for GSK3b binding. In contrast, no interaction was detected between BCL2L12A and GSK3b. In vitro kinase and l-phosphatase assays showed that GSK3b phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12_153–191 fragment directly interrupted GSK3bmediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3b interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.     

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.     

BetaPix-a enhances the activity of phospholipase Cgamma1 by binding SH3 domain in breast cancer

Phospholipase C-gamma1 (PLCgamma1) plays a critical role in cell growth and proliferation by generating the second messengers, diacylglycerol and 1, 4, 5-inositol triphosphate. To investigate the roles of Src homology domain 2 and domain 3 of PLCgamma1 in PLCgamma1-mediated cell signaling, we characterized some proteins binding to these domains in the MCF7 and MDA-MB-231 breast cancer cell lines. Of the several proteins that bind to glutathione-S-transferase-SH2/SH2/SH3, we identified an 85 kDa protein that binds to the SH3 domain of PLCgamma1 as the guanine nucleotide exchange factor, p21-activated protein kinase-interacting exchange factor-a (betaPix-a). BetaPix-a co-immunoprecipitated with PLCgamma1 in breast cancer tissues extracts and in MCF7 and MDA-MB-231 cell extracts. In addition, PDGF-stimulated PLCgamma1 activity was elevated in betaPix-a-overexpressing NIH3T3 cells. Our results suggest that betaPix-a binds to the Src homology domain 3 of PLCgamma1 and promotes tumor growth in breast cancer by enhancing the activity PLCgamma1.