Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

Lactate dehydrogenase A regulates autophagy and tamoxifen resistance in breast cancer

Estrogen receptor (ER) antagonist, tamoxifen has been universally used for the treatment of the ER-positive breast cancer; however, the inevitable emergence of resistance to tamoxifen obstructs the successful treatment of this cancer. So, there is an immediate requirement for the search of a novel therapeutic target for treatment of this cancer. Acquired tamoxifen-resistant breast cancer cell lines MCF-7 (MCF-7/TAM-R) and T47D (T47D/TAM-R) showed higher apoptotic resistance accompanied by induction of pro-survival autophagy compared to their parental cells. Besides, tamoxifen resistance was associated with reduced production of ATP and with overexpression of glycolytic pathways, leading to induced autophagy to meet the energy demand. Further, our study revealed that LDHA; one of the key molecules of glycolysis in association with Beclin-1 induced pro-survival autophagy in tamoxifen-resistant breast cancer. Mechanistically, pharmacological and genetic inhibition of LDHA reduced the pro-survival autophagy, with the restoration of apoptosis and reverting back the EMT like phenomena noticed in tamoxifen-resistant breast cancer. In total, targeting LDHA opened a novel strategy to interrupt autophagy and tamoxifen resistance in breast cancer.     

Enhanced autophagy reveals vulnerability of P-gp mediated epirubicin resistance in triple negative breast cancer cells

Epirubicin (EPI) is widely used for triple negative breast cancer (TNBC), but a substantial number of patients develop EPI resistance that is associated with poor outcome. The underlying mechanism for EPI resistance remains poorly understood. We have developed and characterized an EPI-resistant (EPI-R) cell line from parental MDA-MB-231 cells. These EPI-R cells reached stable growth in the medium containing 8 μg/ml of EPI. They overexpressed P-glycoprotein (P-gp) and contained numerous autophagic vacuoles. The suppression of P-gp overexpression and/or autophagy restored the sensitivity of these EPI-R cells to EPI. We further show that autophagy conferred resistance to EPI on MDA cells by blocking the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated pro-apoptotic signals. Together, these results reveal a synergistic role of P-gp, autophagy, and NF-κB pathways in the development of EPI resistance in TNBC cells. They also suggest that blocking the P-gp overexpression and autophagy may be an effective means of reducing EPI resistance.     

IFNB1/interferon-β-induced autophagy in MCF-7 breast cancer cells counteracts its proapoptotic function

IFNB1/interferon (IFN)-β belongs to the type I IFNs and exerts potent antiproliferative, proapoptotic, antiangiogenic and immunemodulatory functions. Despite the beneficial effects of IFNB1 in experimental breast cancers, clinical translation has been disappointing, possibly due to induction of survival pathways leading to treatment resistance. Defects in autophagy, a conserved cellular degradation pathway, are implicated in numerous cancer diseases. Autophagy is induced in response to cancer therapies and can contribute to treatment resistance. While the type II IFN, IFNG, which in many aspects differs significantly from type I IFNs, can induce autophagy, no such function for any type I IFN has been reported. We show here that IFNB1 induces autophagy in MCF-7, MDAMB231 and SKBR3 breast cancer cells by measuring the turnover of two autophagic markers, MAP1LC3B/LC3 and SQSTM1/p62. The induction of autophagy in MCF-7 cells occurred upstream of the negative regulator of autophagy MTORC1, and autophagosome formation was dependent on the known core autophagy molecule ATG7 and the IFNB1 signaling molecule STAT1. Using siRNA-mediated silencing of several core autophagy molecules and STAT1, we provide evidence that IFNB1 mediates its antiproliferative effects independent of autophagy, while the proapoptotic function of IFNB1 was strongly enhanced in the absence of autophagy. This suggests that autophagy induced by IFNB1 promoted survival, which might contribute to tumor resistance against IFNB1 treatment. It may therefore be clinically relevant to reconcile a role for IFNB1 in the treatment of breast cancer with concomitant inhibition of autophagy.     

MiR-133a Is Functionally Involved in Doxorubicin-Resistance in Breast Cancer Cells MCF-7 via Its Regulation of the Expression of Uncoupling Protein 2

The development of novel targeted therapies holds promise for conquering chemotherapy resistance, which is one of the major hurdles in current breast cancer treatment. Previous studies indicate that mitochondria uncoupling protein 2 (UCP-2) is involved in the development of chemotherapy resistance in colon cancer and lung cancer cells. In the present study we found that lower level of miR133a is accompanied by increased expression of UCP-2 in Doxorubicin-resistant breast cancer cell cline MCF-7/Dox as compared with its parental cell line MCF-7. We postulated that miR133a might play a functional role in the development of Doxorubicin-resistant in breast cancer cells. In this study we showed that: 1) exogenous expression of miR133a in MCF-7/Dox cells can sensitize their reaction to the treatment of Doxorubicin, which is coincided with reduced expression of UCP-2; 2) knockdown of UCP-2 in MCF-7/Dox cells can also sensitize their reaction to the treatment of Doxorubicin; 3) intratumoral delivering of miR133a can restore Doxorubicin treatment response in Doxorubicin-resistant xenografts in vivo, which is concomitant with the decreased expression of UCP-2. These findings provided direct evidences that the miR133a/UCP-2 axis might play an essential role in the development of Doxorubicin-resistance in breast cancer cells, suggesting that the miR133a/UCP-2 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in breast cancer.     

Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.     

BID-deficient breast cancer MCF-7 cells as a model for the study of autophagy in cancer therapy

The BH3-only death factors share just the short BH3 domain with the other Bcl-2 family subclasses. With the exception of BID, which might also bind to BAX, they are thought to act by binding to and neutralizing Bcl-2 like survival factors.(1) Camptothecin (CPT)-induced apoptosis in breast cancer MCF-7 cells is associated with activation of cathepsin B and aggregation of BAX and BID on mitochondria. BID knock down protects cancer cells against apoptosis and induces autophagy, manifested with increased expression of Beclin 1 and MAP1LC3. The compensatory increase in the concentration of Hrk (another member of the BH3-only protein family) and its co-localization with BCL-2 on organelles in BID(-) breast cancer cells has also been observed. Nonetheless, Hrk is not able to substitute for BID in triggering apoptosis. Its role in autophagy induction is also doubtful, since MAP1LC3 expression was equally high in BID(-)Hrk(-) and BID(-)Hrk(+) breast cancer cells exposed to CPT. We conclude that BID can serve as a molecular switch between apoptosis and autophagy. BID(+) and BID(-) breast cancer MCF-7 cells could be considered to be a useful model for the study of the molecular interdependences between apoptosis and autophagy and the role of both processes in cancer therapy.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.     

Differences in the starvation-induced autophagy response in MDA-MB-231 and MCF-7 breast cancer cells

Breast cancer is a heterogeneous disease with distinct subtypes that have made targeted therapy of breast cancer challenging. Previous studies have demonstrated that an altered autophagy capacity can influence the development of breast cancer. However, the molecular differences in starvation-induced autophagic responses in MDA-MB-231 and MCF-7 cells have not been fully elucidated. In this study, we found that an increase of LC3B-II protein expression level and a decrease of the p62 protein expression level in both cells treated by Earle’s balanced salt solution. Meanwhile, we observed an increase of autophagosome using transmission electron microscopy and an enhancement in the green fluorescence intensity of LC3B protein by confocal microscopy. Furthermore, we detected the expression of 13 autophagy-related (ATG) genes and 11 autophagy signaling pathway-related genes using qPCR. Among 13 ATG genes, we found that 6 genes were up-regulated in treated MDA-MB-231 cells, while 4 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. In addition, among 11 autophagy signaling pathway-related genes, 7 genes were up-regulated in treated MDA-MB-231 cells, while 5 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. These findings suggest that the autophagic response to starvation was different in the two treated cell lines, which will contribute to further study on the molecular mechanism of starvation-induced autophagy and improve the targeted therapy of breast cancer.     

BID-Deficient Breast Cancer MCF-7 Cells as a Model for the Study of Autophagy in Cancer Therapy

The BH3-only death factors share just the short BH3 domain with the other Bcl-2 family subclasses. With the exception of BID, which might also bind to BAX, they are thought to act by binding to and neutralizing Bcl-2 like survival factors.Camptothecin (CPT)-induced apoptosis in breast cancer MCF-7 cells is associated with activation of cathepsin B and aggregation of BAX and BID on mitochondria. BID knock down protects cancer cells against apoptosis and induces autophagy, manifested with increased expression of Beclin1 and MAP1LC3. The compensatory increase in the concentration of Hrk (another member of the BH3-only protein family) and its co-localization with BCL-2 on organelles in BID(-) breast cancer cells has also been observed. Nonetheless, Hrk is not able to substitute for BID in triggering apoptosis. Its role in autophagy induction is also doubtful, since MAP1LC3 expression was equally high in BID(-)Hrk(-) and BID(-)Hrk(+) breast cancer cells exposed to CPT. We conclude that BID can serve as a molecular switch between apoptosis and autophagy. BID(+) and BID(-) breast cancer MCF-7 cells could be considered to be a useful model for the study of the molecular interdependences between apoptosis and autophagy and the role of both processes in cancer therapy.

Addenda to:

Cathepsins and BID are Involved in the Molecular Switch between Apoptosis and Autophagy in Breast Cancer MCF-7 Cells Exposed to Camptothecin

M. Lamparska-Przybysz, B. Gajkowska and T. Motyl

J Physiol Pharmacol 2005; 56:159-79     

上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Inhibiting ROS-TFE3-dependent autophagy enhances the therapeutic response to metformin in breast cancer

Autophagy modulation is a potential therapeutic strategy for breast cancer, and a previous study indicated that metformin exhibits significant anti-carcinogenic activity. However, the ability of metformin to induce autophagy and its role in breast cancer cell death remains unclear. In this study, we exposed MCF-7 cells to different concentrations of metformin (2.5, 5, and 10?mM) for 48?h, and metformin-induced significant apoptosis in the MCF-7 cells. The expression levels of CL-PARP (poly(ADP-ribose) polymerase 1) and the ratio of BAX to BCL-2 were significantly increased. In addition to apoptosis, we showed that metformin increased autophagic flux in MCF-7 cells, as evidenced by the upregulation of LC3-II and downregulation of P62/SQSTM1. Moreover, pharmacological or genetic blocking of autophagy increased metformin-induced apoptosis, indicating a cytoprotective role of autophagy in metformin-treated MCF-7 cells. Mechanistically, metformin-induced TFE3^(Ser321) dephosphorylation activated TFE3 nuclear translocation and increased of TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes and, subsequently, initiated autophagy in MCF-7 cells. Importantly, we found that metformin triggered the generation of reactive oxygen species (ROS) in MCF-7 cells. Furthermore, N-acetyl-l-cysteine (NAC), a ROS scavenger, abrogated the effects of metformin on TFE3-dependent autophagy. Notably, TFE3 expression positively correlated with breast cancer development and poor prognosis in patients. Taken together, these data demonstrate that blocking ROS-TFE3-dependent autophagy to enhance the activity of metformin warrants further attention as a treatment strategy for breast cancer.     

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.     

KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway and predicts good prognosis in breast cancer

Tamoxifen resistance represents a daunting challenge to the successful treatment for breast cancer. Krüppel-like factor 4 has critical roles in the development and progression of breast cancer, but its expression, function and regulation in the efficacy of TAM therapy in breast cancer have yet to be investigated. Here, we examined the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 correlated with increased TAM sensitivity in breast cancer cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERα and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness to TAM in T47D and TAM-resistant MCF-7/TAM cells. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion and migration. Moreover, KLF4 expression was down-regulated in breast cancer tumor tissues and high expression of KLF4 was associated with favorable outcomes. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and p38 were more activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors significantly suppressed cell viability. Knockdown of KLF4 activated ERK and p38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and p38 signaling and resulted in increased sensitivity to TAM. Therefore, our findings suggested that KLF4 contributed to TAM sensitivity in breast cancer via phosphorylation modification of ERK and p38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for the TAM-resistant patients.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p