ArticlePDF Available

Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells

Authors:
Rachael McCloy at Garvan Institute of Medical Research
Rachael McCloy
E J Shelley
E J Shelley
E J Shelley
  • This person is not on ResearchGate, or hasn't claimed this research yet.
C G Roberts
C G Roberts
C G Roberts
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Ebru Boslem at EnGeneIC Ltd, Lane Cove, Australia
Ebru Boslem
  • EnGeneIC Ltd, Lane Cove, Australia
Show all 11 authorsHide
Download full-text PDFDownload full-text PDF
Read full-text
Download citation

Abstract and Figures

Background: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. Methods: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. Results: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. Conclusion: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.
The response of 4-OHT-sensitive and -resistant breast cancer cells to both tamoxifen and persin-induced apoptosis. (A) MCF-7/TAM-R and MCF 5-21/23 cells were treated with an apoptotic concentration of 4-OHT (7.5 μM) or vehicle control, and the induction of apoptosis was analysed in attached and floating cells by M30-FITC positivity 24 h after treatment. Values are calculated from at least three independent experiments±s.e. (B) MCF-7/TAM-R and MCF 5-21/23 cells were treated with an apoptotic concentration of persin (10 μg ml−1) or vehicle control, and the induction of apoptosis was analysed in attached and floating cells 24 h after treatment. Values are calculated from at least three independent experiments±s.e. **P<0.005, ***P<0.001, ****P<0.0001 for treated cells vs untreated controls.
The response of 4-OHT-sensitive and -resistant breast cancer cells to both tamoxifen and persin-induced apoptosis. (A) MCF-7/TAM-R and MCF 5-21/23 cells were treated with an apoptotic concentration of 4-OHT (7.5 μM) or vehicle control, and the induction of apoptosis was analysed in attached and floating cells by M30-FITC positivity 24 h after treatment. Values are calculated from at least three independent experiments±s.e. (B) MCF-7/TAM-R and MCF 5-21/23 cells were treated with an apoptotic concentration of persin (10 μg ml−1) or vehicle control, and the induction of apoptosis was analysed in attached and floating cells 24 h after treatment. Values are calculated from at least three independent experiments±s.e. **P<0.005, ***P<0.001, ****P<0.0001 for treated cells vs untreated controls.
… 
Co-treatment with persin and 4-OHT reverses 4-OHT resistance. (A) TAM-R and MCF 5-23 cells were treated with a non-apoptotic concentration of persin (1 μg ml−1) or 4-OHT (7.5 μM) alone or in combination for 24 h, then attached and floating populations were analysed for M30-FITC positivity by flow cytometry. Overlays of representative histograms showing the M30-FITC-positive (apoptotic) fraction. (B) Bars, mean of M30-positive fractions from at least three independent experiments±s.e. **P<0.01, ***P<0.001, for combination treatment vs single treatment.
Co-treatment with persin and 4-OHT reverses 4-OHT resistance. (A) TAM-R and MCF 5-23 cells were treated with a non-apoptotic concentration of persin (1 μg ml−1) or 4-OHT (7.5 μM) alone or in combination for 24 h, then attached and floating populations were analysed for M30-FITC positivity by flow cytometry. Overlays of representative histograms showing the M30-FITC-positive (apoptotic) fraction. (B) Bars, mean of M30-positive fractions from at least three independent experiments±s.e. **P<0.01, ***P<0.001, for combination treatment vs single treatment.
… 
Persin-induced apoptosis is associated with the upregulation and activation of markers of endoplasmic reticulum stress. (A) MCF-7 and T-47D cells were treated with an apoptotic concentration of persin (P; 10 μg ml−1), the endoplasmic reticulum stress inducer, tunicamycin (T; 10 μg ml−1), or untreated (C) for 8, 16, or 24 h, and levels of BiP and CHOP were determined by immunoblotting. β-Actin was used as a loading control. Representative blots from three independent experiments are shown. (B) MCF-7 and T-47D cells were treated with tunicamycin (T; 10 μg ml−1), an apoptotic concentration of persin (P; 10 μg ml−1) or vehicle control (C) for 8, 16, or 24 h. RNA was then collected, cDNA produced, XBP-1 cDNA amplified using PCR, and the product digested with the restriction enzyme PstI to determine the proportion of undigested (active) to digested (inactive) XBP-1. Representative gels from at least three independent experiments are shown. (C) Densitometric analysis of undigested vs digested XBP-1 PCR product in MCF-7 and T-47D cells treated with tunicamycin (10 μg ml−1) or persin (10 μg ml−1) for 8, 16, or 24 h, and vehicle controls. Bars, mean of three independent experiments±s.e. *P<0.05 for treated cells vs untreated controls.
Persin-induced apoptosis is associated with the upregulation and activation of markers of endoplasmic reticulum stress. (A) MCF-7 and T-47D cells were treated with an apoptotic concentration of persin (P; 10 μg ml−1), the endoplasmic reticulum stress inducer, tunicamycin (T; 10 μg ml−1), or untreated (C) for 8, 16, or 24 h, and levels of BiP and CHOP were determined by immunoblotting. β-Actin was used as a loading control. Representative blots from three independent experiments are shown. (B) MCF-7 and T-47D cells were treated with tunicamycin (T; 10 μg ml−1), an apoptotic concentration of persin (P; 10 μg ml−1) or vehicle control (C) for 8, 16, or 24 h. RNA was then collected, cDNA produced, XBP-1 cDNA amplified using PCR, and the product digested with the restriction enzyme PstI to determine the proportion of undigested (active) to digested (inactive) XBP-1. Representative gels from at least three independent experiments are shown. (C) Densitometric analysis of undigested vs digested XBP-1 PCR product in MCF-7 and T-47D cells treated with tunicamycin (10 μg ml−1) or persin (10 μg ml−1) for 8, 16, or 24 h, and vehicle controls. Bars, mean of three independent experiments±s.e. *P<0.05 for treated cells vs untreated controls.
… 
The role of CHOP in the induction of persin-induced apoptosis. (A) MCF-7 cells were treated for 24 h with CHOP-specific siRNAs (siCHOP; 20 nM), non-targeting controls (siNT; 20 nM) or mock transfected and then treated for a further 24 h with an apoptotic concentration of persin (P; 10 μg ml−1) or vehicle control (C). Protein samples were prepared and analysed for CHOP expression by immunoblotting. Representative blots from four independent experiments are shown. β-Actin was used as a loading control. (B) Attached and floating populations were then analysed for M30-FITC positivity by flow cytometry. Bars, mean of M30-positive fractions from four independent experiments±s.e. **P<0.005 for CHOP siRNA treatment vs NT siRNA treatment.
The role of CHOP in the induction of persin-induced apoptosis. (A) MCF-7 cells were treated for 24 h with CHOP-specific siRNAs (siCHOP; 20 nM), non-targeting controls (siNT; 20 nM) or mock transfected and then treated for a further 24 h with an apoptotic concentration of persin (P; 10 μg ml−1) or vehicle control (C). Protein samples were prepared and analysed for CHOP expression by immunoblotting. Representative blots from four independent experiments are shown. β-Actin was used as a loading control. (B) Attached and floating populations were then analysed for M30-FITC positivity by flow cytometry. Bars, mean of M30-positive fractions from four independent experiments±s.e. **P<0.005 for CHOP siRNA treatment vs NT siRNA treatment.
… 
Inhibition of caspase-4 reduces persin-induced apoptosis. (A) MCF-7 and T-47D cells were treated with an apoptotic concentration of persin (10 μg ml−1) alone or in combination with caspase-4 inhibitor (20 μM) or pan-caspase inhibitor (20 μM) for 24 h, and then attached and floating populations were analysed for M30-FITC positivity by flow cytometry. Representative histograms showing the M30-FITC-positive (apoptotic) fraction are shown. (B) Bars, mean of M30-positive fractions from three independent experiments±s.e. ***P<0.001; ****P<0.0001 for combination treatment vs single treatment.
+1
Inhibition of caspase-4 reduces persin-induced apoptosis. (A) MCF-7 and T-47D cells were treated with an apoptotic concentration of persin (10 μg ml−1) alone or in combination with caspase-4 inhibitor (20 μM) or pan-caspase inhibitor (20 μM) for 24 h, and then attached and floating populations were analysed for M30-FITC positivity by flow cytometry. Representative histograms showing the M30-FITC-positive (apoptotic) fraction are shown. (B) Bars, mean of M30-positive fractions from three independent experiments±s.e. ***P<0.001; ****P<0.0001 for combination treatment vs single treatment.
… 
Figures - available via license: Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported
Content may be subject to copyright.
bjc201369
3a.pdf
Content uploaded by Ebru Boslem
Author content
All content in this area was uploaded by Ebru Boslem on Aug 14, 2014
Content may be subject to copyright.
Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cel
ls.pdf
Available via license: CC BY-NC-SA 3.0
Content may be subject to copyright.
A preview of the PDF is not available
... CHOP is a best-characterized factor in the transition from ER stress to apoptosis [30][31]. Our previous study demonstrated that CHOP plays a vital role in kaempferolinduced apoptosis via ER stress pathway [12]. ...
... CHOP is the best-characterized factor in the transition from ER stress to apoptosis [30][31]. To examine the contribution of CHOP in kaempferol-induced cell death, we treated HepG2 cells with ER stress inhibitor, 4-PBA, or transfected HepG2 cells with CHOP siRNA and found that 4-PBA pretreatment or CHOP siRNA decreased the protein levels of Atg5, Atg7, Beclin1 and LC3-II, and attenuated kaempferol-induced autophagy. ...
Kaempferol induces hepatocellular carcinoma cell death via endoplasmic reticulum stress-CHOP-autophagy signaling pathway
Article
Full-text available
  • Jul 2017
Kaempferol is a flavonoid compound that has gained widespread attention due to its antitumor functions. However, the underlying mechanisms are still not clear. The present study investigated the effect of kaempferol on hepatocellular carcinoma and its underlying mechanisms. Kaempferol induced autophagy in a concentration- and time-dependent manner in HepG2 or Huh7 cells, which was evidenced by the significant increase of autophagy-related genes. Inhibition of autophagy pathway, through 3-methyladenine or Atg7 siRNA, strongly diminished kaempferol-induced apoptosis. We further hypothesized that kaempferol can induce autophagy via endoplasmic reticulum (ER) stress pathway. Indeed, blocking ER stress by 4-phenyl butyric acid (4-PBA) or knockdown of CCAAT/enhancer-binding protein homologous protein (CHOP) with siRNA alleviated kaempferol-induced HepG2 or Huh7 cells autophagy; while transfection with plasmid overexpressing CHOP reversed the effect of 4-PBA on kaempferol-induced autophagy. Our results demonstrated that kaempferol induced hepatocarcinoma cell death via ER stress and CHOP-autophagy signaling pathway; kaempferol may be used as a potential chemopreventive agent for patients with hepatocellular carcinoma.
View
... The combined use of persin and 4-OHT on TAM-R cells showed a synergistic induction of drug-resistant cell apoptosis pre-dominantly mediated by the CHOP-dependent endoplasmic reticulum stress (ERS) signaling cascades (McCloy et al., 2013). Jaeumkanghwa soup (JEKHT) is a compound composed of 12 traditional herbs (Jung et al., 2010). ...
Interactions Between Natural Products and Tamoxifen in Breast Cancer: A Comprehensive Literature Review
Article
Full-text available
  • Jun 2022
Introduction: Tamoxifen (TAM) is the most commonly used hormone therapeutic drug for the treatment of estrogen receptor-positive (ER+) breast cancer. 30%–70% of clinical breast cancer patients use natural products, which may increase the likelihood of drug interactions. Objective: To evaluate the evidence for the interactions between natural products and TAM in breast cancer. Methods: Electronic databases, including PubMed, CINAHL Plus ( via EbscoHost), European PMC, Medline, and Google Scholar, were searched for relevant publications. The search terms include complementary and alternative medicine, natural products, plant products, herbs, interactions, tamoxifen, breast cancer, and their combinations. Results: Various in vitro and in vivo studies demonstrated that the combined use of natural products with TAM produced synergistic anti-cancer effects, including improved inhibition of tumor cell growth and TAM sensitivity and reduced side effects or toxicity of TAM. In contrast, some natural products, including Angelica sinensis (Oliv.) Diels [Apiaceae] , Paeonia lactiflora Pall . , Rehmannia glutinosa (Gaertn.) DC. , Astragalus mongholicus Bunge, and Glycyrrhiza glabra L. [Fabaceae], showed estrogen-like activity, which may reduce the anti-cancer effect of TAM. Some natural products, including morin, silybin, epigallocatechin gallate (EGCG), myricetin, baicalein, curcumin, kaempferol, or quercetin, were found to increase the bioavailability of TAM and its metabolites in vivo . However, three are limited clinical studies on the combination of natural products and TAM. Conclusion: There is evidence for potential interactions of various natural products with TAM in pre-clinical studies, although the relevant clinical evidence is still lacking. Further studies are warranted to evaluate the potential interactions of natural products with TAM in clinical settings.
View
... Nec-1 had no effect on cell viability at all concentrations tested, whereas Z-VAD reduced cell viability at concentrations exceeding 250 μM (Fig. 1A, B). According to previously published concentrations and assay times from the literature and our own viability data presented here, pretreating PIG1 cells with 20 μM Z-VAD and 40 μM Nec-1 for 1 h was selected in the following assay [21,22]. Oxidative stress was induced by the treatment with 500 mM hydrogen peroxide (H 2 O 2 ) as previously described [3]. ...
Oxeiptosis: a novel pathway of melanocytes death in response to oxidative stress in vitiligo
Article
Full-text available
  • Feb 2022
Vitiligo is a cutaneous depigmenting autoimmune disease caused by the extensive destruction of epidermal melanocytes. Convincing data has defined a critical role for oxidative stress in the pathogenesis of vitiligo. Oxeiptosis is a caspase-independent cell death modality that was reportedly triggered by oxidative stress and operative in pathogen clearance. However, whether oxeiptosis exists in oxidative stress-induced melanocytes demise in vitiligo remains undetermined. In the present study, we initially found that other cell death modalities might exist in addition to the well-recognized apoptosis and necroptosis in H2O2-treated melanocytes. Furthermore, AIFM1 was found to be dephosphorylated at Ser116 in oxidative stress-induced melanocytes death, which was specific to oxeiptosis. Moreover, KEAP1 and PGAM5, upstream of the AIFM1 in oxeiptosis, were found to operate in melanocytic death. Subsequently, the KEAP1-PGAM5-AIFM1 signaling pathway was proved to be involved in oxidative stress-triggered melanocytes demise through the depletion of KEAP1 and PGAM5. Altogether, our study indicated that oxeiptosis might occur in melanocytes death under oxidative stress and contribute to the pathogenesis of vitiligo.
View
... As evident from the literature, acetylsalicylic acid (aspirin) is a tamoxifen sensitizer and was shown to reverse resistance elevating UPR/EnR stress (34). The avocado-derived toxin persin also augments the proapoptotic response of drug-resistant breast cancer cells to Tam by enhancing EnR stress signaling (35). Inhibition of PDE4D using either siRNAs or pharmacologic inhibitors restored tamoxifen sensitivity by activating EnR stress. ...
HBXIP is a novel regulator of the unfolded protein response that sustains tamoxifen resistance in ER+ breast cancer
Article
Full-text available
  • Jan 2022
  • J BIOL CHEM
Endocrine therapy-resistant estrogen receptor-positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug-resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifen (TAM)-induced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation (ERAD), enhances UPR-element (UPRE) reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a (IRE1α) and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, TAM treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase (PERK) and activating transcription factor 6α (ATF6α). Clinical investigations and Kaplan-Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a novel regulator of EnR homeostasis and a potential target for TamR breast cancer therapy.
View
... In this line, we demonstrated that combination of tamoxifen with a specific inhibitor of the phosphodiesterase 4D elevates cyclic AMP (cAMP) levels, inducing EnR stress and activation of stress-related kinases p38 and JNK, leading to apoptotic cell death and tamoxifen sensitization [138]. Moreover, a plant toxin, persin was shown to overcome tamoxifen resistance by induction of EnR stress response characterized by increased levels of CHOP, the EnR chaperone-binding immunoglobulin protein (Bip), and spliced X-box binding protein 1 (XBP-1), leading to apoptotic cell death [139]. Similar to UPR, induction of autophagy may also be either pro-survival or pro-apoptotic depending on the source and duration of the stressor that ultimately dictates cell fate. ...
Endocrine resistance in breast cancer: from molecular mechanisms to therapeutic strategies
Article
Full-text available
  • Oct 2021
  • J MOL MED-JMM
Estrogen receptor-positive (ER +) breast cancer accounts for approximately 75% of all breast cancers. Endocrine therapies, including selective ER modulators (SERMs), aromatase inhibitors (AIs), and selective ER down-regulators (SERDs) provide substantial clinical benefit by reducing the risk of disease recurrence and mortality. However, resistance to endocrine therapies represents a major challenge, limiting the success of ER + breast cancer treatment. Mechanisms of endocrine resistance involve alterations in ER signaling via modulation of ER (e.g., ER downregulation, ESR1 mutations or fusions); alterations in ER coactivators/corepressors, transcription factors (TFs), nuclear receptors and epigenetic modulators; regulation of signaling pathways; modulation of cell cycle regulators; stress signaling; and alterations in tumor microenvironment, nutrient stress, and metabolic regulation. Current therapeutic strategies to improve outcome of endocrine-resistant patients in clinics include inhibitors against mechanistic target of rapamycin (mTOR), cyclin-dependent kinase (CDK) 4/6, and the phosphoinositide 3-kinase (PI3K) subunit, p110α. Preclinical studies reveal novel therapeutic targets, some of which are currently tested in clinical trials as single agents or in combination with endocrine therapies, such as ER partial agonists, ER proteolysis targeting chimeras (PROTACs), next-generation SERDs, AKT inhibitors, epidermal growth factor receptor 1 and 2 (EGFR/HER2) dual inhibitors, HER2 targeting antibody-drug conjugates (ADCs) and histone deacetylase (HDAC) inhibitors. In this review, we summarize the established and emerging mechanisms of endocrine resistance, alterations during metastatic recurrence, and discuss the approved therapies and ongoing clinical trials testing the combination of novel targeted therapies with endocrine therapy in endocrine-resistant ER + breast cancer patients.
View
... The UPR signal consists of stressors located on the ER membrane: IRE1, XBP-1, PERK (RNA-dependent protein kinase-like ER kinase), and ATF-6 (Donadelli et al., 2011;Lin et al., 2019). Also, CHOP represents the center-point for the three ER stress transducers listed (PERK, IRE1α, and ATF-6) and is the most characterized mediator in the transformation of ER stress to apoptosis (Marhfour et al., 2012;McCloy et al., 2013). However, when the ER function is severely impaired, the organelle elicits cell death signals through activation of CHOP, which in turn is described to promote apoptosis by B-cell lymphoma gene-2 (Bcl-2)-like protein 11 (BIM) induction and Bcl-2 inhibition (Donadelli et al., 2011;Lin et al., 2019). ...
Crosstalk between ER-stress and apoptosis in irradiated HepG2 cells with gemcitabine: implication of PI3K/AKT and IκB/NF-κB signaling pathways
Article
Full-text available
  • Jan 2020
Gemcitabine (GEM) is an anti-cancer drug that has been used in the treatment of a wide range of human cancers. However, cancer cells developing resistance to GEM which may limit its use. So, adjuvant therapies either enhance the effects of GEM or overcome resistance are needed for the treatment of cancer. In this study, we examined the influence of the interaction between γ-radiation (RAD) and GEM against HepG2 cells acquired resistance to GEM. Based on our data, the combination therapy elevates ROS production, induces endoplasmic reticulum (ER)-stress response machinery, up-regulates caspase-3 and p53 as well as boosts PTEN proficiency which blocked PI3K/AKT pathway that inhibits IκB/NF-κB signaling. It could be concluded that therapeutic strategies of gemcitabine in cancer might be potentiated by radiotherapy to reach better therapeutic outcomes.
View
MC3T3-E1 cells lead to bone loss in Staphylococcus aureus osteomyelitis through oxeiptosis pathway
Preprint
Full-text available
  • Mar 2024
Bone loss is a significant complication of Staphylococcus aureus osteomyelitis, for which there is currently no efficacious treatment despite research indicating the involvement of various mechanisms in bone loss during bone infections. Oxeiptosis, a caspase independent form of cell death induced by oxidative stress, has been implicated in pathogen elimination. Nevertheless, the role of oxeiptosis in the extinction of osteoblasts during S. aureus infection remains uncertain. In the current investigation, preliminary evidence suggests the existence of alternative mechanisms of cell demise beyond the established pathways of apoptosis and necrosis following S. aureus infection in osteoblasts. Moreover, our findings indicate that dephosphorylation of AIFM1 at Ser116 occurs during S. aureus infection in osteoblasts, ultimately resulting in osteoblast death. Additionally, KEAP1 and PGAM5, situated upstream of AIFM1 in this cascade, are implicated in the process of osteoblast death. Our study demonstrates that the KEAP1-PGAM5-AIFM1 signaling pathway is implicated in S. aureus-induced osteoblast death through the depletion of KEAP1 and PGAM5. This suggests that infection through the oxeiptosis pathway may contribute to osteoblast death and subsequent bone loss in individuals with osteomyelitis.
View
Avocado ( Persea americana Mill) and its phytoconstituents: potential for cancer prevention and intervention
Article
  • Oct 2023
Dietary compounds, including fruits, vegetables, nuts, and spices, have been shown to exhibit anticancer properties due to their high concentrations of vitamins, minerals, fiber, and secondary metabolites, known as phytochemicals. Although emerging studies suggest that avocado (Persea americana Mill) displays antineoplastic properties in addition to numerous other health benefits, current literature lacks an updated comprehensive systematic review dedicated to the anticancer effects of avocado. This review aims to explore the cancer-preventive effects of avocados and the underlying molecular mechanisms. The in vitro studies suggest the various avocado-derived products and phytochemicals induced cytotoxicity, reduced cell viability, and inhibited cell proliferation. The in vivo studies revealed reduction in tumor number, size, and volume as well. The clinical studies demonstrated that avocado leaf extract increased free oxygen radical formation in larynx carcinoma tissue. Various avocado products and phytochemicals from the avocado fruit, including avocatin-B, persin, and PaDef defensin, may serve as viable cancer prevention and treatment options based on current literature. Despite many favorable outcomes, past research has been limited in scope, and more extensive and mechanism-based in vivo and randomized clinical studies should be performed before avocado-derived bioactive phytochemicals can be developed as cancer preventive agents.
View
Aberrant fatty acid profile and FFAR4 signaling confer endocrine resistance in breast cancer
Article
Full-text available
  • Dec 2019
  • J EXP CLIN CANC RES
Background Evidence suggests that fatty acid receptor FFAR4 plays a tumor-promoting role in adipose tissue-adjacent malignancies, but its clinical relevance remains unexplored. Here, we investigated the clinical significance and underlying mechanisms of FFAR4 in hormone receptor-positive breast cancer (HRPBC). Methods FFAR4 expression was assessed by immunohistochemistry in an exploration cohort of 307 breast cancer cases collected from two independent institutes. Two public breast cancer microarray datasets served as validation cohorts. Gas chromatography-mass spectrometry was employed to identify FFAR4 ligands in normal and cancerous breast tissues. Survival analyses were performed in all cohorts and designated molecular subgroups. Mechanistic studies were performed in vitro in hormone receptor-positive breast cancer cell lines MCF-7 and T-47D. Results Aberrant FFAR4 expression and endogenous FFAR4 ligands were identified in breast cancer tissues, five FFAR4 ligands showed significantly elevated proportions in cancerous versus normal tissues. In the exploration cohort, FFAR4 was demonstrated as an independent prognostic factor for recurrences (HR: 2.183, 95% CI: 1.360–3.504, P = 0.001) and breast cancer-specific deaths (HR: 2.102, 95% CI: 1.173–3.766, P = 0.013) in HRPBC cases. In contrast, FFAR4 expression was not associated with prognosis in hormone receptor-negative cases. In the validation cohorts, FFAR4 mRNA levels were also observed to be associated with disease recurrence in estrogen receptor-positive cases, but not so in estrogen receptor-negative cases. FFAR4 activation by endogenous ligands and a synthetic ligand TUG891 significantly dampened tamoxifen’s efficacy on HRPBC cells, whereas FFAR4 knockdown or antagonist AH7614 abrogated this effect. Furthermore, FFAR4-induced tamoxifen resistance was dependent on ERK and AKT pathways in HRPBC. Conclusions Our results establish a novel role of FFAR4 and its ligands in the complicated interactions between tissue lipid profile and cancer biology. FFAR4 signaling confers tamoxifen resistance in HRPBC cell line and FFAR4 expression can serve as a prognostic biomarker for tamoxifen-treated HRPBC patients. FFAR4 may serve as a potential target for anti-breast cancer therapies, especially in endocrine resistant cases. Electronic supplementary material The online version of this article (10.1186/s13046-019-1040-3) contains supplementary material, which is available to authorized users.
View
Tamoxifen-induced cytotoxicity in breast cancer cells is mediated by glucose-regulated protein 78 (GRP78) via AKT (Thr308) regulation
Article
  • Jun 2016
Glucose regulated protein 78 (GRP78) has recently been suggested to be associated with drug resistance in breast cancer patients. However, the precise role of GRP78 in drug resistance and the involved signaling pathways are not clearly understood. In the present study, we show that among a panel of drugs, namely Paclitaxel (TAX), Doxorubicin (DOX), 5-fluorouracil (5-FU), UCN-01 and Tamoxifen (TAM) used, TAM alone up-regulated the expression of GRP78 significantly and induced apoptosis in MCF-7 and MDA-MB-231 cells. Interestingly, inhibition of GRP78 by a specific pharmacological inhibitor, VER-155008 augmented TAM-induced apoptosis, and overexpression of GRP78 rendered the cells resistant to TAM-induced cell death suggesting a role for GRP78 in TAM-induced cytotoxicity. Mechanistically, the expression of phosphorylated AKT as determined by Western blot analyses revealed that TAM selectively upregulated phosphorylation of AKT at Thr308 but not at Ser473, and siRNA silencing of GRP78 resulted in inhibition of AKT phosphorylation at Thr308 but not at Ser473. Further, TAM induced phosphorylation of GSK3β, a downstream substrate of AKT, and GRP78 inhibitor, VER155008 inhibited TAM-induced GSK3β phosphorylation. These results, thus suggests a role for GRP78 in TAM-induced AKT activation. Additionally, co-localization studies by immunofluorescence, and immunoprecipitation experiments demonstrated a complex formation of AKT and GRP78. Furthermore, in glucose-free medium, the cells were sensitized to TAM-induced cell death that was associated with reduced AKT phosphorylation at Thr308, thus strengthening the association of AKT regulation with drug response. Collectively, our findings identify a role of GRP78 in AKT regulation in response to TAM in breast cancer cells.
View
Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer
Article
Full-text available
  • Nov 2011
Adjuvant treatment with tamoxifen substantially improves survival of women with estrogen-receptor positive (ER+) tumors. Tamoxifen resistance (TAMR) limits clinical benefit. RRR-α-tocopherol ether-linked acetic acid analogue (α-TEA) is a small bioactive lipid with potent anticancer activity. We evaluated the ability of α-TEA in the presence of tamoxifen to circumvent TAMR in human breast cancer cell lines. Two genotypically matched sets of TAM-sensitive (TAMS) and TAM-resistant (TAMR) human breast cancer cell lines were assessed for signal-transduction events with Western blotting, apoptosis induction with Annexin V-FITC/PI assays, and characterization of cholesterol-rich microdomains with fluorescence staining. Critical involvement of selected mediators was determined by using RNA interference and chemical inhibitors. Growth-factor receptors (total and phosphorylated forms of HER-1 and HER-2), their downstream prosurvival mediators pAkt, pmTOR, and pERK1/2, phosphorylated form of estrogen receptor-α (pER-α at Ser-167 and Ser-118, and cholesterol-rich lipid microdomains were highly amplified in TAMR cell lines and enhanced by treatment with TAM. α-TEA disrupted cholesterol-rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators, and induced DR5-mediated mitochondria-dependent apoptosis via an endoplasmic reticulum stress-triggered pro-death pJNK/CHOP/DR5 amplification loop. Furthermore, methyl-β-cyclodextrin (MβCD), a chemical disruptor of cholesterol rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators and to induce apoptosis. Data for the first time document that targeting cholesterol-rich lipid microdomains is a potential strategy to circumvent TAMR, and the combination of α-TEA + TAM can circumvent TAMR by suppression of prosurvival signaling via disruption of cholesterol-rich lipid microdomains and activation of apoptotic pathways via induction of endoplasmic reticulum stress.
View
Synthesis and in vitro evaluation of analogues of avocado-produced toxin (+)-(R)-persin in human breast cancer cells
Article
Full-text available
  • Oct 2011
A structure-activity study of several new synthetic analogues of the avocado-produced toxin persin has been conducted, with compounds being evaluated for their cytostatic and pro-apoptotic effects in human breast cancer cells. A 4-pyridinyl derivative demonstrated activity comparable to that of the natural product, suggesting future directions for exploration of structure-activity relationships.
View
Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time
Article
Full-text available
  • Nov 2011
  • P NATL ACAD SCI USA
In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.
View
ChemInform Abstract: New Drugs, Old Fashioned Ways: ER Stress Induced Cell Death
Article
Full-text available
  • Aug 2012
  • CURR PHARM BIOTECHNO
Discovery of small molecules able to induce several cellular self-killing mechanisms improved cancer therapy in the last years. Research focused on canonical apoptotic (mitochondria or death receptor related) pathways to induce cell death in several hematologic and solid malignancies, showing that treatment with different synthetic and natural compounds reactivates the cell death machinery previously silenced in resistant cancer cells. Besides the canonical apoptotic pathways, alternative pathways of cell death induction have recently been rediscovered as potential new targets for cancer therapy. Under certain conditions, protein folding can be disturbed causing an accumulation of unfolded proteins inside the endoplasmic reticulum (ER). This situation leads to stress ER, involving the transcriptional and translational machinery to induce the expression and post-transcriptional modifications of many factors involved in ER stress response mediated cell death. In this scenario, some apoptotic players like caspase 4 or caspase 12 start to control cell fate by inducing downstream cell death proteins. Recently, inhibitors of protein deacetylases have been demonstrated to potently induce this alternative cell death pathway and will be reviewed here.
View
Br??ning A, Friese K, Burges A, Mylonas ITamoxifen enhances the cytotoxic effects of nelfinavir in breast cancer cells. Breast Cancer Res 12(4): R45
Article
Full-text available
  • Jul 2010
The HIV protease inhibitor nelfinavir is currently under investigation as a new anti-cancer drug. Several studies have shown that nelfinavir induces cell cycle arrest, endoplasmic reticulum stress, autophagy, and apoptosis in cancer cells. In the present article, the effect of nelfinavir on human breast cancer cells is examined and potential combination treatments are investigated. The effects of nelfinavir and tamoxifen on the human breast cancer cell lines MCF7, T47 D, MDA-MB-453, and MDA-MB-435 were tested by analysing their influence on cell viability (via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay), apoptosis (annexin binding, poly(ADP-ribose) polymerase cleavage), autophagy (autophagy marker light chain 3B expression), endoplasmic reticulum stress (binding protein and activating transcription factor 3 expression), and the occurrence of oxidative stress (intracellular glutathione level). Nelfinavir induced apoptosis in all four breast cancer cell lines tested, although the extent of autophagy and endoplasmic reticulum stress varied among the cell lines. The concentration of nelfinavir needed for an efficient induction of apoptosis in breast cancer cells could be reduced from 15 μg/ml to 6 μg/ml when combined with tamoxifen. At a concentration of 6 μg/ml, tamoxifen substantially enhanced the endoplasmic reticulum stress reaction in those cell lines that responded to nelfinavir with binding protein (BiP) upregulation (MCF7, T47D), and enhanced autophagy in cell lines that responded to nelfinavir treatment with autophagy marker light chain 3B upregulation (MDA-MB-453). Although tamoxifen has been described to be able to induce oxidative stress at concentrations similar to those applied in this study (6 μg/ml), we observed that nelfinavir but not tamoxifen reduced the intracellular glutathione level of breast cancer cells within hours of application by up to 32%, suggesting the induction of oxidative stress was an early event and an additional cause of the apoptosis induced by nelfinavir. The results demonstrate that nelfinavir may be an effective drug against breast cancer and could be combined with tamoxifen to enhance its efficacy against breast cancer cells. Moreover, the cytotoxic effect of a tamoxifen and nelfinavir combination was independent of the oestrogen receptor status of the analysed breast cancer cells, suggesting a potential benefit of a combination of these two drugs even in patients with no hormone-responsive tumours. We therefore recommend that clinical studies on nelfinavir with breast cancer patients should include this drug combination to analyse the therapeutic efficacy as well as the safety and tolerability of this potential treatment option.
View
View
Circumvention of tamoxifen resistance by the pure anti-estrogen ICI 182, 780
Article
Both primary and acquired resistance to the growth-inhibitory effects of anti-estrogens (e.g., tamoxifen) limits the clinical usefulness of these drugs in the treatment of breast cancer. The new, steroidal anti-estrogen ICI 182,780 was tested for its ability to inhibit the proliferation of a tamoxifen-resistant variant of the parental MCF-7 human breast-cancer cell line. Two cell lines cloned from the MCF-7 line were used for these experiments: a tamoxifen-sensitive line, MCF 5-21, and a tamoxifen-resistant line, MCF 5-23. Compared with tamoxifen, ICI 182,780 appeared to be 150 and 1540 times more effective in inhibiting cell growth in the 5-21 and 5-23 sub-lines respectively. ICI 182,780 completely circumvented tamoxifen resistance at a concentration of (5 to 10) × 10−9 M in this model. Based on IC50 concentrations, the 5-23 line was 22-fold more resistant to tamoxifen than the 5-21 line, but only 2-fold more resistant to ICI 182, 780, reducing relative resistance by 10-fold in the resistant line. There were no differences in ER parameters between the 2 lines. ER numbers/cell were: 40500 and 34800 and the KD 0.48 and 0.15 × 10−9 M in the 5-21 and 5-23 cells respectively. In the 5-23 cells, the concentrations of ICI 182, 780 and tamoxifen resulting in a 50% inhibition of 3 H-estradiol binding were 2.3 × 10−8 M and 1 × 10−6 M, respectively (cf. estradiol 0.89 × 10−9 M). Thus, one potential mechanism for the increased effectiveness of ICI 182,780 may relate to the increased affinity of this drug for the estrogen receptor as compared with tamoxifen.
View
Gamma linolenic acid with tamoxifen as primary therapy in breast cancer
Article
Gamma linolenic acid (GLA) has been proposed as a valuable new cancer therapy having selective anti-tumour properties with negligible systemic toxicity. Proposed mechanisms of action include modulation of steroid hormone receptors. We have investigated the effects of GLA with primary hormone therapy in an endocrine-sensitive cancer. Thirty-eight breast cancer patients (20 elderly Stage I-II, 14 locally advanced, 4 metastatic) took 8 capsules of oral GLA/day (total = 2.8 g) in addition to tamoxifen 20 mg od (T+GLA). Quality and duration of response were compared with matched controls receiving tamoxifen 20 mg od alone (n = 47). Serial tumour biopsies were taken to assess changes in oestrogen receptor (ER) and bcl-2 expression during treatment. GLA was well tolerated with no major side effects. T+GLA cases achieved a significantly faster clinical response (objective response vs. static disease) than tamoxifen controls, evident by 6 weeks on treatment (p = 0.010). There was significant reduction in ER expression in both treatment arms with T+GLA objective responders sustaining greater ER fall than tamoxifen counterparts (6-week biopsy p = 0.026; 6-month biopsy p = 0.019). We propose GLA as a useful adjunct to primary tamoxifen in endocrine-sensitive breast cancer. The effects of GLA on ER function and the apparent enhancement of tamoxifen-induced ER down-regulation by GLA require further investigation. Int. J. Cancer 85:643–648, 2000. © 2000 Wiley-Liss, Inc.
View
Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions
Article
  • Jan 2000
The 78kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER–) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER– breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER– tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy.
View
Targeting apoptotic and autophagic pathways for cancer therapeutics
Article
  • Oct 2010
Apoptosis and autophagy are genetically regulated, evolutionarily-conserved processes that regulate cell fate; however, apoptosis invariably contributes to cancer cell death, whereas autophagy plays the Janus role of cancer cell survival and death. In this review, we firstly focus on targeting cancer cells via several key apoptotic pathways for anti-cancer therapy. Additionally, we demonstrate that some autophagic pathways play dual roles in cancer, and further elucidate the intricate relationship between apoptosis and autophagy. In summary, these findings may ultimately allow biologists to harness apoptotic and autophagic pathways as novel targets for cancer therapeutics.
View