Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

LncRNA MEG3 regulates breast cancer proliferation and apoptosis through miR-141-3p/RBMS3 axis

Maternally expressed 3 (MEG3) and RNA binding motif single stranded interacting protein 3 (RBMS3) are abnormally expressed in breast cancer susceptibility genes (BRCA), but the mechanism of the two in breast cancer (BC) is unclear. By performing in vivo and in vitro experiments, we found that MEG3 and RBMS3 were low-expressed, negatively correlated with high-expressed miR-141-3p, were positively correlated with each other in BC. MEG3 targeted miR-141-3p, and miR-141-3p targeted RBMS3. MEG3, which was mainly distributed in BC cytoplasm, could down-regulate miR-141-3p and up-regulate RBMS3, and reverse effect of miR-141-3p on related gene expressions and on promoting cancer development. Overexpressed MEG3 inhibited growth of xenografts, promoted cell apoptosis via regulating apoptosis related factors, and up-regulated RBMS3 expression but down-regulated miR-141-3p. The findings of this study showed that MEG3 inhibited proliferation and promoted apoptosis of BC cells through the miR-141-3p/RBMS3 axis, and MEG3 inhibited growth of xenografts through miR-141-3p.     

Inhibition of PKM2 sensitizes triple-negative breast cancer cells to doxorubicin

Cancer cells alter regular metabolic pathways in order to sustain rapid proliferation. One example of metabolic remodeling in cancerous tissue is the upregulation of pyruvate kinase isoenzyme M2 (PKM2), which is involved in aerobic glycolysis. Indeed, PKM2 has previously been identified as a tumor biomarker and as a potential target for cancer therapy. Here, we examined the effects of combined treatment with doxorubicin and anti-PKM2 small interfering RNA (siRNA) on triple-negative breast cancer (TNBC). The suppression of PKM2 resulted in changes in glucose metabolism, leading to decreased synthesis of adenosine triphosphate (ATP). Reduced levels of ATP resulted in the intracellular accumulation of doxorubicin, consequently enhancing the therapeutic efficacy of this drug in several triple-negative breast cancer cell lines. Furthermore, the combined effect of PKM2 siRNA and doxorubicin was evaluated in an in vivo MDA-MB-231 orthotopic breast cancer model. The siRNA was systemically administered through a polyethylenimine (PEI)-based delivery system that has been extensively used. We demonstrate that the combination treatment showed superior anticancer efficacy as compared to doxorubicin alone. These findings suggest that targeting PKM2 can increase the efficacy of chemotherapy, potentially providing a new approach for improving the outcome of chemotherapy in patients with TNBC.     

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

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

Ramentaceone,a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells

The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer.     

Dual VEGFR-2/PIM-1 kinase inhibition towards surmounting the resistance to antiangiogenic agents via hybrid pyridine and thienopyridine-based scaffolds: Design,synthesis and biological evaluation

Angiogenesis is a hallmark in cancer. Most antiangiogenic agents block the action of vascular endothelial growth factor (VEGF). In clinic, patients develop hypoxia-mediated resistance consistent with vascular responses to these agents. Recent studies underlying such resistance revealed hypoxia-inducible PIM-1 kinase upregulation which promotes cancer progression. PIM-1 kinase expression is thus viewed as a new resistance mechanism to antiangiogenic agents. Hence, combining PIM kinase inhibitors with anti-VEGF therapies provides synergistic antitumor response. Inspired by these facts, the current study aims at designing novel dual VEGFR-2/PIM-1 kinase inhibitors via molecular hybridization and repositioning of their pharmacophoric features. Moreover, enhancing the cytotoxic potential of the designed compounds was considered via incorporating moieties mimicking caspase 3/7 activators. Accordingly, series of novel pyridine and thieno[2,3-b]pyridine derivatives were synthesized and screened via MTT assay for cytotoxic activities against normal fibroblasts and four cancer cell lines (HepG-2, Caco-2, MCF-7 and PC-3). Compounds 3a, 9e, 10b and 10c exhibited anticancer activities at nanomolar IC₅₀ with promising safety, activated caspase 3/7 and induced apoptosis as well as DNA fragmentation more than doxorubicin in the four cancer cell lines. Furthermore, they exerted promising dual VEGFR-2/PIM-1 kinase inhibition and significantly exhibited higher therapeutic potential to alter the expression levels of VEGF, p53 and cyclin D than doxorubicin. Interestingly, the most active anticancer compound 10b conferred the highest dual VEGFR-2/PIM-1 kinase inhibition. Finally, their in silico ligand efficiency metrics were acceptable.     

Genistein induces Ca2+ -mediated, calpain/caspase-12-dependent apoptosis in breast cancer cells

Genistein, a soy-derived isoflavone, has been suggested for breast cancer prevention; however, use of soy products for this purpose remains controversial. Genistein has been reported to regulate growth of tumor cells, although the involved molecular mechanisms are not defined. Here we report that genistein induces apoptosis in breast cancer cells via activation of the Ca2+ -dependent proapoptotic proteases, mu-calpain, and caspase-12. The treatment of MCF-7 breast cancer cells with genistein induced a sustained increase in concentration of intracellular Ca2+ resulting from depletion of the endoplasmic reticulum Ca2+ stores. This increase in Ca2+ was associated with activation of mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 substrates and antibodies to active (cleaved) forms of the enzymes. Selective inhibition of Ca2+ binding sites of mu-calpain, forced increase of the cytosolic Ca2+ buffering capacity, and caspase inhibition decreased apoptotic indices in the genistein-treated cells. Our results suggest that Ca2+ -dependent proteases are potential targets for genistein in breast cancer cells and that the cellular Ca2+ regulatory activity of genistein underlies its apoptotic mechanism.     

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.     

Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

Corema album Leaves Mediate DNA Damage in Triple-Negative Breast Cancer Cells

Corema (C.) album is a shrub endemic to the Atlantic coast and has been described as yielding beneficial effects for human health. Nevertheless, studies concerning the bioactivity of C. album leaves are scarce. This study aims at investigating the anticancer potential and mode of action, of an hydroethanolic extract of C. album leaves (ECAL) on triple-negative breast cancer. This is a poor survival breast cancer subtype, owing to its high risk of distant reappearance, metastasis rates and the probability of relapse. The ECAL ability to prevent tumor progression through (i) the inhibition of cell proliferation (cell viability); (ii) the induction of apoptosis (morphological changes, TUNEL assay, caspase-3 cleaved) and (iii) the induction of DNA damage (PARP1 and γH2AX) with (iv) the involvement of NF-κB and of ERK1/2 pathways (AlphaScreen assay) was evaluated. ECAL activated the apoptotic pathway (through caspase-3) along with the inhibition of ERK and NF-κB pathways causing DNA damage and cell death. The large polyphenolic content of ECAL was presumed to be accountable for these effects. The extract of C. album leaves can target multiple pathways and, thus, can block more than one possible means of disease progression, evidencing the anticancer therapeutic potential from a plant source.     

RBMS3 at 3p24 Inhibits Nasopharyngeal Carcinoma Development via Inhibiting Cell Proliferation, Angiogenesis, and Inducing Apoptosis

Deletion of the short arm of chromosome 3 is one of the most frequent genetic alterations in many solid tumors including nasopharyngeal carcinoma (NPC), suggesting the existence of one or more tumor suppressor genes (TSGs) within the frequently deleted region. A putative TSG RBMS3 (RNA binding motif, single stranded interacting protein 3), located at 3p24-p23, has been identified in our previous study. Here, we reported that downregulation of RBMS3 was detected in 3/3 NPC cell lines and 13/15 (86.7%) primary NPC tissues. Functional studies using both overexpression and suppression systems demonstrated that RBMS3 has a strong tumor suppressive role in NPC. The tumor suppressive mechanism of RBMS3 was associated with its role in cell cycle arrest at the G1/S checkpoint by upregulating p53 and p21, downregulating cyclin E and CDK2, and the subsequent inhibition of Rb-ser780. Further analysis demonstrated that RBMS3 had a pro-apoptotic role in a mitochondrial-dependent manner via activation of caspase-9 and PARP. Finally, RBMS3 inhibited microvessel formation, which may be mediated by down-regulation of MMP2 and β-catenin and inactivation of its downstream targets, including cyclin-D1, c-Myc, MMP7, and MMP9. Taken together, our findings define a function for RBMS3 as an important tumor suppressor gene in NPC.     

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.     

CircSLC39A8 attenuates paclitaxel resistance in ovarian cancer by regulating the miR‑185‑5p/BMF axis

Chemoresistance to paclitaxel (PTX) is one of the main reasons for treatment failure and poor prognosis in patients with advanced ovarian cancer. Therefore, it is imperative to explore the mechanisms related to chemotherapy resistance in ovarian cancer to find potential therapeutic targets. Circular RNAs (circRNAs) play important roles in cancer development and progression. However, their biological functions and clinical significance in ovarian cancer have not been fully elucidated. Therefore, in this study, we aimed to investigate the function and underlying mechanism of hsa_circ_0002782 (circSLC39A8), identified by circRNA sequencing, in regulating PTX resistance. The effects of circSLC39A8 on PTX resistance was assessed by cell viability, colony formation, flow cytometry assays and an in vivo subcutaneous xenografted tumor mouse model. RNA immunoprecipitation and dual-luciferase reporter assays were performed to verify the interaction between circSLC39A8 and the miR-185–5p/BMF signal axis. We found that circSLC39A8 was downregulated in PTX-resistant ovarian cancer cells and tissues, and its low expression was associated with poor prognosis. Biologically, circSLC39A8 knockdown promoted PTX resistance in vitro and in vivo, while circSLC39A8 overexpression showed the opposite effect. Mechanistically, circSLC39A8, acting as an endogenous sponge for miR-185–5p, could relieve the inhibition of miR-185–5p on the expression of its downstream target, BMF; thus enhancing the sensitivity of ovarian cancer to PTX. Our findings demonstrate that circSLC39A8 can promote PTX sensitivity by regulating the miR-185–5p/BMF axis. This may be a valuable prognostic biomarker and a promising therapeutic target for patients with ovarian cancer.     

β1-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters

Molecular interactions of tumor cells with the microenvironment are regarded as onset of chemotherapy resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate a mechanism of CAM-DR in breast cancer cells in vitro. We show that human MCF-7 and MDA-MB-231 breast cancer cells decrease their sensitivity towards cisplatin, doxorubicin, and mitoxantrone cytotoxicity upon binding to collagen type 1 (COL1) or fibronectin (FN). The intracellular concentrations of doxorubicin and mitoxantrone were decreased upon cell cultivation on COL1, while cellular cisplatin levels remained unaffected. Since doxorubicin and mitoxantrone are transporter substrates, this refers to ATP binding cassette (ABC) efflux transporter activities. The activation of the transporters BCRP, P-gp and MRP1 was shown by fluorescence assays to distinguish the individual input of these transporters to resistance in presence of COL1 and related to their expression levels by western blot. An ABC transporter inhibitor was able to re-sensitize COL1-treated cells for doxorubicin and mitoxantrone toxicity. Antibody-blocking of β₁-integrin (ITGB1) induced sensitization towards the indicated cytostatic drugs by attenuating the increased ABC efflux activity. This refers to a key role of ITGB1 for matrix binding and subsequent transporter activation. A downregulation of α₂β₁ integrin following COL1 binding appears as clear indication for the relationship between ITGB1 and ABC transporters in regulating resistance formation, while knockdown of ITGB1 leads to a significant upregulation of all three transporters. Our data provide evidence for a role of CAM-DR in breast cancer via an ITGB1 – transporter axis and offer promising therapeutic targets for cancer sensitization.     

The Role of Kif4A in Doxorubicin-Induced Apoptosis in Breast Cancer Cells

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.     

Down-regulation of thiamine transporter THTR2 gene expression in breast cancer and its association with resistance to apoptosis

The recent molecular identification of two thiamine transporters, SLC19A2 (THTR1) and SLC19A3 (THTR2), has provided the opportunity to study thiamine transporter gene expression in human malignancies. We compared RNA levels of both THTR1 and THTR2 in a panel of human breast tumors and corresponding normal tissues. THTR2 RNA levels were down-regulated in breast cancer to 14% of the level found in corresponding normal tissues, while THTR1 levels were unchanged. Both thiamine transport genes were cloned and expressed in a breast cancer cell line to examine the impact of reconstituted thiamine transport gene expression on drug and radiation sensitivity and on resistance to apoptosis. THTR2-transfected breast cancer cells showed a 2.5-fold increase in specific THTR2 activity and a 3-fold increase in cytotoxicity against a bromoacetyl ester derivative of thiamine. Surprisingly, these cells also showed a 3-fold increase in sensitivity to doxorubicin and an increase in sensitivity to ionizing radiation, but no change in sensitivity to methotrexate or paclitaxel. TUNEL assays demonstrate an increase in apoptosis in THTR2-transfected cells exposed to doxorubicin and radiation, and Western blot analysis suggests that apoptosis associated with these cytotoxic stresses is mediated at least in part by a caspase-3-dependent pathway. Therefore, thiamine transporter THTR2 gene expression is down-regulated in breast cancer, which may contribute to resistance to apoptosis in these tumors.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

Biochemical and Structural Insights into Carbonic Anhydrase XII/Fab6A10 Complex

6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer.