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.     

Bmi-1稳定干扰增加乳腺癌细胞对化疗药物的敏感性

目的:观察Bmi-1稳定干扰后对乳腺癌细胞药敏性的影响.方法:构建Bmi-1 shRNA逆转录病毒表达载体,并用该逆转录病毒感染人乳腺癌细胞,建立Bmi-1稳定干扰的乳腺癌细胞株.采用不同浓度的化疗药物5-氟尿嘧啶和顺铂处理上述细胞,MTT法观察乳腺癌细胞的生长抑制率.结果:免疫印迹检测结果表明Bmi-1稳定干扰的乳腺癌细胞株成功建立.MTT检测显示Bmi-1稳定干扰的乳腺癌细胞(MDA-MB-435S-Bmi-1/RNAi)增殖抑制率显著高于对照细胞(MDA-MB-435S-vector)(P<0.05),并且呈浓度依赖性.其中在MDA-MB-435S-vector中5-氟尿嘧啶和顺铂的IC50值分别为1.7233 mg/L和2.9065 mg/L,而在MDA-MB-435S Bmi-1/RNAi稳定细胞株5-氟尿嘧啶和顺铂中的IC50值分别为0.9733 mg/L和1.8563mg/L(P<0.05).结论:Bmi-1在乳腺癌细胞对化疗药物敏感性中发挥重要作用,Bmi-1干扰能增加乳腺癌细胞对化疗药物的敏感性.     

2DG enhances the susceptibility of breast cancer cells to doxorubicin

2DG causes cytotoxicity in cancer cells by disrupting thiol metabolism while Doxorubicin (DOX) induces cytotoxicity in tumor cells by generating reactive oxygen species (ROS). Here we examined the combined cytotoxic action of 2DG and DOX in rapidly dividing T47D breast cancer cells vs. slowly growing MCF-7 breast cancer cells. T47D cells exposed to the combination of 2DG/DOX significantly decreased cell survival compared to controls, while 2DG/DOX had no effect on MCF-7 cells. 2DG/DOX also disrupted the oxidant status of T47D treated cells, decreased intracellular total glutathione and increased glutathione disulfide (%GSSG) compared to MCF-7 cells. Lipid peroxidation increased in T47D cells treated with 2DG and/or DOX, but not in MCF-7 cells. T47D cells were significantly protected by NAC, indicating that the combined treatment exerts its action by increasing ROS production and disrupting antioxidant stores. When we inhibited glutathione synthesis with BSO, T47D cells became more sensitive to 2DG/DOX-induced cytotoxicity, but NAC significantly reversed this cytotoxic effect. Finally, 2DG/DOX, and BSO significantly increased the %GSSG in T47D cells, an effect which was also reversed by NAC. Our results suggest that exposure of rapidly dividing breast cancer cells to 2DG/DOX enhances cytotoxicity via oxidative stress and via disruptions to thiol metabolism.     

Epithelium removal alters responsiveness of guinea pig trachea to substance P

Removal of epithelium from mammalian tracheae has been shown to enhance responsiveness to a variety of contractile and relaxant agents. One of the most dramatic shifts reported has been for guinea pig tracheal tissue denuded of epithelium and treated with substance P. We investigated whether this shift in responsiveness was because of 1) removal of an epithelium-associated enzyme, neutral endopeptidase, which degrades substance P and 2) loss of an epithelium-derived noncyclooxygenase relaxant factor. Using a muscle bath preparation we performed concentration-response curves with substance P and acetylcholine on indomethacin-treated tissues with and without intact epithelium and with and without pretreatment with the neutral endopeptidase inhibitor, phosphoramidon. Epithelium removal potentiated the mean agonist concentration calculated to causes 30% of the maximal contractile response by 148-fold for substance P and by 7-fold for acetylcholine. Phosphoramidon potentiated the contractile response to substance P, but not to acetylcholine, by both the epithelium-intact and denuded tissues (P less than 0.05). However, the degree of enhancement by phosphoramidon was much greater in the intact tissues. With phosphoramidon treatment, therefore, the difference in responsiveness to substance P between the intact and denuded tissues was reduced from 148-fold to 18-fold. This effect of phosphoramidon suggests that the hyperresponsiveness to substance P of epithelium-denuded airway tissue is largely because of removal of neutral endopeptidase. Because all tissues were treated with indomethacin, the leftward shifts in substance P and in acetylcholine responsiveness induced by epithelium removal further suggest that an epithelium-derived noncyclooxygenase factor other than neutral endopeptidase also modulates the contractile response to substance P and to acetylcholine.     

Effects of substance P on experimental parkinsonian syndrome

The aim of this study was to investigate the effect of substance P (SP) on parkinsonian syndrome and the generator of pathologically enhanced excitation (GPEE) in the caudate nuclei (CN). Repeated i. p. administration of MPTP in 12 month rats induced oligokinesia and rigidity followed by the high amplitude slow and rapid waves in both CN. The changes of electrical activity in CN were more prominent than in the sensorimotor cortex. The bilateral intracaudate injection of SP (5 micrograms) resulted in an increase in motor activity and almost completely abolished the rigidity. The reduction of extrapyramidal symptoms was considered as a result of the inhibition of GPEE. The changes of the SP balance in nigro-striatal system was suggested to be one of the pathogenetic links of parkinsonian syndrome.     

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca clearance from MCF-7 breast cancer cells

The expression of the plasma membrane Ca²⁺ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca²⁺ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression – either by treatment or overexpression – led to enhanced Ca²⁺ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca²⁺ homeostasis. These results suggest that modulation of Ca²⁺ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.     

Anti-inflammatory agents and substance P depletion in experimental ileitis

To understand the interactions between substance P and gut inflammation, changes in substance P levels were evaluated in a chronic model of ileitis in response to three anti-inflammatory agents with distinct mechanisms of action. The agents were the prostaglandin E(1) analogue misoprostol (30 mug/kg, s.c., b.i.d.), the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mug/ml in drinking water) and the leumedin, N-(fluorenyl-9-methoxycarbonyl)-L-leucine (NPC 15199, 10 mg/kg, s.c.). Ileitis was induced by a transmural injection of trinitrobenzene sulphonic acid (TNBS 30 mg/kg in 50% ethanol) into the distal ileum of guinea-pigs. All anti-inflammatory therapies were introduced after TNBS administration and continued until day 7, when guinea-pigs were killed. Ileal substance P levels were measured by radioimmunoassay, and granulocyte infiltration was quantified by myeloperoxidase (MPO) activity. Protein and nitrite (an index of nitric oxide formation) levels in a luminal saline lavage were quantified in all groups. TNBS ileitis caused a marked reduction in ileal substance P content and increased MPO activity, protein and nitrite secretion. The nitric oxide synthase inhibitor, L-NAME, completely restored all parameters to baseline. Misoprostol attenuated the granulocyte infiltration and exacerbated protein leak but had no effect on substance P levels. In contrast, NPC 15199 had no effect on granulocyte infiltration but normalized substance P levels and protein leak. Only L-NAME and NPC 15199 blocked the TNBS induced increase in nitrite levels. These results suggest that the regulation of granulocyte infiltration in this model is unrelated to changes in substance P levels. Inhibition of nitric oxide synthase was the most effective therapeutic strategy in TNBS ileitis but the precise interactions between nitric oxide and the enteric nervous system during inflammatory states remain to be defined.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

miR‐200c enhances radiosensitivity of human breast cancer cells

Due to the intrinsic resistance of many tumors to radiotherapy, current methods to improve the survival of cancer patients largely depend on increasing tumor radiosensitivity. It is well‐known that miR‐200c inhibits epithelial–mesenchymal transition (EMT), and enhances cancer cell chemosensitivity. We sought to clarify the effects of miR‐200c on the radiosensitization of human breast cancer cells. In this study, we found that low levels of miR‐200c expression correlated with radiotolerance in breast cancer cells. miR‐200c overexpression could increase radiosensitivity in breast cancer cells by inhibiting cell proliferation, and by increasing apoptosis and DNA double‐strand breaks. Additionally, we found that miR‐200c directly targeted TANK‐binding kinase 1 (TBK1). However, overexpression of TBK1 partially rescued miR‐200c mediated apoptosis induced by ionizing radiation. In summary, miR‐200c can be a potential target for enhancing the effect of radiation treatment on breast cancer cells. J. Cell. Biochem. 114: 606–615, 2013. © 2012 Wiley Periodicals, Inc.     

Curcumin enhances TRAIL-induced apoptosis of breast cancer cells by regulating apoptosis-related proteins

The TNF-related apoptosis inducing ligand (TRAIL) has promising anti-cancer therapeutic activity, although significant percentage of primary tumors resistant to TRAIL-induced apoptosis remains an obstacle to the extensive use of TRAIL-based mono-therapies. Natural compound curcumin could potentially sensitize resistant cancer cells to TRAIL. We found that the combination of TRAIL with curcumin can synergistically induces apoptosis in three TRAIL-resistant breast cancer cell lines. The mechanism behind this synergistic cell death was investigated by examining an effect of curcumin on the expression and activation of TRAIL-associated cell death proteins. Immunoblotting, RNA interference, and use of chemical inhibitors of TRAIL-activate signaling revealed differential effects of curcumin on the expression of Mcl-1 and activities of ERK and Akt. Curcumin-induced production of reactive oxygen species did not affect total expression of DR5 but it enhanced mobilization of DR5 to the plasma membrane. In these breast cancer cells curcumin also induced downregulation of IAP proteins. Taken together, our data suggest that a combination of TRAIL and curcumin is a potentially promising treatment for breast cancer, although the specific mechanisms involved in this sensitization could differ even among breast cancer cells of different origins.     

Evaluation of serum level of substance P and tissue distribution of NK-1 receptor in colorectal cancer

Colorectal cancer (CRC) is known as the most common form of malignancies in the world and its occurrence is annually increasing. Due to the relatively high death rates in patients, finding better diagnostic and prognostic factors are required. Substance P (SP) belongs to the tachykinin family that acts as an immunomodulator by binding to the neurokinin-1 receptor (NK1R). The interaction of SP with NK1R might be involved in tumor cell proliferation, angiogenesis, and migration. Hence, this study was aimed to evaluate the serum SP level and tissue distribution of NK1Rs in CRC. Also, we assessed the relationship between tissue distribution of NK1R and some different tumor characteristics, including tumor size, and lymph node status. Recruiting 38 patients primarily diagnosed with CRC, the tissue distribution of NK1R was immunohistochemically evaluated in tumor tissues and their adjacent normal tissue. The serum level of SP was measured using an ELISA method in both cases and healthy control group. The SP value was significantly increased in the serum of patients in comparison with the healthy group (p?=?0.001). Tumor tissues expressed a higher number of NK1R than adjacent normal tissues (p?=?0.01) considering both the percentage of stained cells and intensity of staining. However, there was not any statistically significant relevance between NK1R distribution and tumor characteristics. The SP/NK1R system is involved in tumorigenesis of CRC, and might be suggested as a potent prognostic or diagnostic factor, or a new target in the treatment of CRC.

     

Hepatocyte growth factor enhances CXCR4 expression favoring breast cancer cell invasiveness

Microenvironmental factors affect different aspects of tumor cell biology, including cell survival, invasion, and metastasis. Here, we report that hepatocyte growth factor and hypoxia may contribute to breast carcinoma cell invasiveness by inducing the chemokine receptor CXCR4. Hepatocyte growth factor enhanced CXCR4 mRNA and protein expression exclusively in MCF-7 (low invasive) carcinoma cells, while in response to hypoxia, CXCR4 induction was observed in both MCF-7 and MDA-MB 231 (highly invasive) carcinoma cells. The receptor induction had a functional role in cancer cells, as demonstrated by the fact that hepatocyte growth factor pretreatment promoted MCF-7 cell migration toward the CXCR4-specific ligand CXCL12. Extracellular signal-regulated protein kinase 1/2 (ERK1/2) and phosphoinositide-3-kinase (PI3K) transduction pathways seemed to be differently implicated in the early induction of CXCR4 by hepatocyte growth factor or hypoxia in the two breast carcinoma cells examined.     

Butyrate activates the monocarboxylate transporter MCT4 expression in breast cancer cells and enhances the antitumor activity of 3-bromopyruvate

Most malignant tumors exhibit the Warburg effect, which consists in increased glycolysis rates with production of lactate, even in the presence of oxygen. Monocarboxylate transporters (MCTs), maintain these glycolytic rates, by mediating the influx and/or efflux of lactate and are overexpressed in several cancer cell types. The lactate and pyruvate analogue 3-bromopyruvate (3-BP) is an inhibitor of the energy metabolism, which has been proposed as a specific antitumor agent. In the present study, we aimed at determining the effect of 3-BP in breast cancer cells and evaluated the putative role of MCTs on this effect. Our results showed that the three breast cancer cell lines used presented different sensitivities to 3-BP: ZR-75-1 ER (+)>MCF-7 ER (+)>SK-BR-3 ER (-). We also demonstrated that 3-BP reduced lactate production, induced cell morphological alterations and increased apoptosis. The effect of 3-BP appears to be cytotoxic rather than cytostatic, as a continued decrease in cell viability was observed after removal of 3-BP. We showed that pre-incubation with butyrate enhanced significantly 3-BP cytotoxicity, especially in the most resistant breast cancer cell line, SK-BR-3. We observed that butyrate treatment induced localization of MCT1 in the plasma membrane as well as overexpression of MCT4 and its chaperone CD147. Our results thus indicate that butyrate pre-treatment potentiates the effect of 3-BP, most probably by increasing the rates of 3-BP transport through MCT1/4. This study supports the potential use of butyrate as adjuvant of 3-BP in the treatment of breast cancer resistant cells, namely ER (-).     

Mullerian inhibiting substance promotes interferon gamma-induced gene expression and apoptosis in breast cancer cells

This report demonstrates that in addition to interferons and cytokines, members of the TGF beta superfamily such as Mullerian inhibiting substance (MIS) and activin A also regulate IRF-1 expression. MIS induced IRF-1 expression in the mammary glands of mice in vivo and in breast cancer cells in vitro and stimulation of IRF-1 by MIS was dependent on activation of the NF kappa B pathway. In the rat mammary gland, IRF-1 expression gradually decreased during pregnancy and lactation but increased at involution. In breast cancer, the IRF-1 protein was absent in 13% of tumors tested compared with matched normal glands. Consistent with its growth suppressive activity, expression of IRF-1 in breast cancer cells induced apoptosis. Treatment of breast cancer cells with MIS and interferon gamma (IFN-gamma) co-stimulated IRF-1 and CEACAM1 expression and synergistic induction of CEACAM1 by a combination of MIS and IFN-gamma was impaired by antisense IRF-1 expression. Furthermore, a combination of IFN-gamma and MIS inhibited the growth of breast cancer cells to a greater extent than either one alone. Both reagents alone significantly decreased the fraction of cells in the S-phase of the cell cycle, an effect not enhanced when they were used in combination. However, MIS promoted IFN-gamma-induced apoptosis demonstrating a functional interaction between these two classes of signaling molecules in regulation of breast cancer cell growth.     

P53 mediated regulation of metallothionein transcription in breast cancer cells

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.     

Degradation of substance P by neurones and glial cells

Neuronal and astroblast-rich cultures from rat brain degrade exogenously added substance P. The rate of degradation is decreased by diisopropylfluorophosphate, phosphoramidon and bacitracin, but not by N-ethylmaleimide or bestatin. When diisopropylfluorophosphate, phosphoramidon and bacitracin are simultaneously present in the culture medium, the degradation of substance P is completely inhibited. These results indicate that the hydrolysis of substance P by intact cells is catalyzed by the post-proline dipeptidylaminopeptidase (EC 3.4.14.5), the thermolysin-like metallopeptidase ("enkephalinase", EC 3.4.24.11) and a yet uncharacterized bacitracin-sensitive activity. While the thermolysin-like metallopeptidase is mainly associated with glial cells, the specific activity of the other enzymes is five times higher in the neuronal culture.