Insulin-Like Growth Factor Binding Protein-3 Induces Senescence by Inhibiting Telomerase Activity in MCF-7 Breast Cancer Cells

doi:10.21203/rs.3.rs-2081030/v1

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Insulin-Like Growth Factor Binding Protein-3 Induces Senescence by Inhibiting Telomerase Activity in MCF-7 Breast Cancer Cells

https://doi.org/10.21203/rs.3.rs-2081030/v1

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published 30 May, 2023

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Insulin-like growth factor binding protein-3 (IGFBP-3) has been known to inhibit the proliferation of various cell types in an insulin-like growth factor (IGF)-independent manner. In this study, we aimed to show that IGFBP-3 induces cellular senescence via suppression of telomerase activity, thereby inhibiting cancer cell proliferation. We found that the induction of IGFBP-3 in MCF-7 cells inhibited cell proliferation. Flow cytometry revealed that the percentage of non-cycling cells was higher in IGFBP-3-expressing cells than in controls. Induction of IGFBP-3 also resulted in morphological changes, such as a flattened cytoplasm and increased granularity, suggesting that IGFBP-3 induces senescence-like phenotype. The percentage of cells containing senescence-associated β-galactosidase activity was 3.3 times higher in IGFBP-3 expressing cells compared to control cells. Telomeric repeat amplification and real-time PCR showed that IGFBP-3 decreased telomerase activity by decreasing the expression of the RNA component (hTR) and catalytic protein component with reverse transcriptase activity (hTERT) of telomerase. These results suggest that IGFBP-3 functions as a negative regulator of breast cancer cell growth by inducing a senescence through the inhibition of telomerase activity.

insulin-like growth factor binding protein-3

insulin-like growth factor I

cell aging

telomerase

breast neoplasm

Insulin-like growth factor binding protein (IGFBP-3) is a multifunctional protein involved in essential cellular processes in various cell systems, including proliferation, differentiation, survival, migration, senescence, autophagy, and angiogenesis^1–3. IGFBP-3 is known to modulate the actions of insulin-like growth factors (IGFs) by regulating their access to the IGF-I receptor, thereby inhibiting or enhancing the activity of IGF^4,5. Additionally, IGFBP-3 exerts many IGF-independent actions, which are mediated by binding to a number of non-IGF proteins and interactions with other growth factors^1–3. IGFBP-3 has been reported to have both tumor suppressing or promoting effects depending on the cell types, cellular environments, and status of coupling with other cell signaling systems³. However, IGFBP-3 acts mainly as a tumor suppressor, which is supported by findings that the disruption of IGFBP-3 at transcriptional and post-translational levels has been implicated in the pathogenesis of some types of cancer including breast, prostate, and lung cancers⁶. Increasing evidence suggests that IGFBP-3 interacts with a variety of proteins or signaling cascades involved in apoptosis and cell cycle regulation^7–12. IGFBP-3 has been reported to inhibit cell proliferation through the induction of apoptosis by increasing the ratio of pro-apoptotic to anti-apoptotic proteins¹³ or by activating caspases¹⁴. In addition, IGFBP-3 may inhibit cell proliferation by modulating the cell cycle. IGFBP-3 has been shown to induce cell cycle arrest through the decrease of p34cdc2, a protein critically involved in cell cycle arrest¹⁵; the increase of the cyclin-dependent kinase (CDK) inhibitory protein, p21/WAF1¹⁶; and the decrease of cell cycle-regulating proteins, such as cyclin D1, cyclin D3, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2, CDK4, and total and phospho-pRb¹⁷. The observed anti-proliferative and pro-apoptotic functions of IGFBP-3 may shed light on its importance on the pathophysiology and etiology of various human diseases, such as cancer, diabetes, vascular disease, asthma and growth disorders^18–20. However, the mechanism for the growth inhibitory action of IGFBP-3 in various cell systems still remains unclear.

Interestingly, previous studies have demonstrated that IGFBP-3 may associate with cellular senescence in many cell types including fibroblasts²¹, epithelial cells²², and endothelial cells^23,24. It has been further identified that the IGFBP-3 enhancer element, a transcriptional regulatory element identified in the 5ʹ untranslated region of the IGFBP-3 gene, activates IGFBP-3 expression in senescent fibroblasts²⁵. Moreover, ectopic expression of IGFBP-3 in young human umbilical vein endothelial cell induces a senescence response, suggesting that IGFBP-3 may be a rate-limiting regulator of endothelial cell senescence²⁴. In breast cancer cells, IGFBP-3 has also been identified as a secreted mediator of cancer cell senescence upon chemotherapeutic drug treatment using a secretome proteomics approach²⁶.

Cellular senescence is also associated with telomere length and telomerase activity. The length of telomeres shortens during differentiation due to the downregulation of telomerase activity in somatic cells, but not in germline cells. As a result, proliferating cells lose about 50–200 bp of telomeric DNA after each replication. This telomere shortening sets a molecular clock for the proliferation capacity of normal cells. In culture, telomerase-negative normal human cells enter a telomere length-dependent growth arrest, which is defined as replicative senescence. In addition, diverse stressful stimuli can induce a senescence-like response known as stress-induced premature senescence. Therefore, senescence is thought to function as an additional barrier against the accumulation of damaged DNA and transformation of cells, as well as an underlying cause of aging²⁷. Telomerase activity is the major maintenance mechanism of telomere in eukaryotes²⁸. Telomerase activity can be detected in early human development, but it is downregulated in most adult tissues²⁹. Although telomerase activity is undetectable in most human tissues, cells from fetal tissue, stem cells, and some types of germ cells and epithelial cells maintain weak, yet detectable, telomerase activity³⁰. In contrast, telomerase activity has been observed in the vast majority of human tumor cell lines^31,32.

In this study, we demonstrate that IGFBP-3 inhibits cell proliferation through a senescence by inhibiting telomerase activity in MCF-7 human breast cancer cells. Furthermore, we elucidate the potential mechanism by which IGFBP-3 alters the expression of telomerase components, such as human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT).

2.1. IGFBP-3 Inhibits Proliferation of MCF-7 Cells

We added ponasterone A to MCF-7-derived cell line to express endogenous IGFBP-3 in an inducible manner. IGFBP-3 concentrations in culture medium were increased on day 1, 2, 3, and 5 with the addition of ponasterone A (Fig. 1A). Co-treatment with ponasterone A and siRNA:IGFBP-3 prevented the increased expression of IGFBP-3 at all the indicated times (Fig. 1A). The induced expression of IGFBP-3 by ponasterone A resulted in a significant inhibition of cell proliferation, ranging from 41–49% on day 3 and 5, when compared to cells without ponasterone A (p < 0.05, p < 0.05, respectively) (Fig. 1B). This inhibitory effect was abolished by co-treatment with siRNA:IGFBP-3, but not scramble siRNA, demonstrating that the effect of IGFBP-3 on cell proliferation is specific (Fig. 1B).

2.2. The Inhibitory Effect of IGFBP-3 Is Mediated by a Mechanism Other Than Apoptosis

Previous studies have reported that IGFBP-3 induces apoptosis in various cell systems^10–14. To determine whether only apoptosis contributed to the inhibition of cell proliferation by IGFBP-3, we performed cell proliferation assays using the pan-caspase inhibitor, z-VAD-fmk. The expression of IGFBP-3 or the addition of TNF-α (30 ng/mL), which is known to activate the caspase system, resulted in significant inhibition of cell proliferation compared to control cells (62% and 73% on day 3, p < 0.05 and p < 0.01, respectively) (Fig. 1C). The inhibitory effect of TNF-α was abolished by co-treatment with z-VAD-fmk (p < 0.05), while the inhibitory effect of IGFBP-3 was not totally abolished by z-VAD-fmk, suggesting that IGFBP-3 inhibited cell proliferation by a mechanism other than caspase-mediated apoptosis.

2.3. IGFBP-3 Increases the Fraction of Non-cycling Cells

Our previous study demonstrated that IGFBP-3 inhibits cell proliferation through the induction of G1 cell cycle arrest in MCF-7 cells¹⁷. Next, to determine whether IGFBP-3 induces senescence, which is a permanent exit from the cell cycle, we looked for an increase in non-cycling cell numbers in cells ectopically expressing IGFBP-3 using flow cytometry (Fig. 2A). The percentage of non-cycling cells was increased from 1.25% (in the absence of ponasterone A) to 4.8% (in the presence of ponasterone A) (p < 0.01) (Fig. 2B). The increase in the percentage of non-cycling cells induced by IGFBP-3 was reduced from 4.8–2.13% by co-treatment with siRNA:IGFBP-3, but not scramble siRNA, suggesting that this effect was specifically due to the increased expression of IGFBP-3. These results indicate that the induced expression of IGFBP-3 increases the fraction of non-cycling cells in this cell system, suggesting IGFBP-3 induces senescence.

2.4. IGFBP-3 Induces Senescence-like Phenotype

The induction of IGFBP-3 resulted in morphological changes, such as a flattened cytoplasm and increased granularity (Fig. 3), which were similar to those reported in senescent cells³⁸. These findings suggest that IGFBP-3 expression induces a senescence-like phenotype. These morphological changes were abolished by siRNA:IGFBP-3, but not z-VAD-fmk or scramble siRNA, suggesting that induction of a senescence-like phenotype by IGFBP-3 is specific, and apoptosis is not the only mechanism responsible for reduced proliferation in cells ectopically expressing IGFBP-3.

2.5. Senescence-associated ß-galactosidase Activity Is Increased by IGFBP-3

We determined the percentage of cells containing SA-ß-galactosidase by immunocytochemistry, which is a marker of senescence³⁷. The percentage of cells containing SA-ß-galactosidase activity was 3.3 times higher in IGFBP-3-expressing cells than in control cells on day 3 (p < 0.01), indicating that IGFBP-3 induced senescence (Fig. 4). Addition of siRNA:IGFBP-3 into the IGFBP-3-expressing cells decreased the percentage of cells containing SA-ß-galactosidase activity about 43% on day 3 (p < 0.01). The increased SA-ß-galactosidase activity by IGFBP-3 was abolished by siRNA:IGFBP-3, but not z-VAD-fmk or scramble siRNA, suggesting that the induction of senescence by IGFBP-3 is specific, and that apoptosis is not responsible for the reduced proliferation observed in these cells.

2.6. IGFBP-3 Decreases Telomerase Activity

Next, we used a TRAP Photometric Enzyme Immunoassay to determine whether IGFBP-3 induced senescence by modulating telomerase activity. The induction of IGFBP-3 significantly decreased telomerase activity compared to cells without ponasterone A (p < 0.01) (Fig. 5), suggesting that decreased telomerase activity may induce senescence in MCF-7 cells. The decrease in telomerase activity by IGFBP-3 was abolished by co-treatment with siRNA:IGFBP-3, but not z-VAD-fmk or scramble siRNA. These results suggest that the modulation of telomerase activity by IGFBP-3 is specific and independent of caspase-mediated apoptosis.

2.7. IGFBP-3 Decreases Reverse Transcriptase Components and RNA of Telomerase

We determined the expression of reverse transcriptase (hTERT) and RNA (hTR) to elucidate the potential mechanism by which IGFBP-3 alters telomerase activity. In reverse-transcription quantitative PCR, IGFBP-3 and GGG-IGFBP-3 (an IGFBP-3 mutant that does not bind IGF-I) transfections in MCF-7 cells decreased the mRNA levels of hTERT and hTR (Fig. 6A and 6B). The addition of siRNA:IGFBP-3 to the IGFBP-3-transfected cells did not decrease the mRNA levels of hTERT and hTR, while the addition of scramble siRNA decreased these mRNA levels (Fig. 6A and 6B). In addition, we determined the mRNA and protein levels of hTERT after addition of exogenous IGFBP-3 at various concentrations in hTERT-overexpressed normal breast cells. Exogenous IGFBP-3 decreased the expression levels of hTERT mRNA (Fig. 6C) and protein (Fig. 6D) in a dose-dependent manner.

In the present study, we demonstrated that IGFBP-3 inhibited cell proliferation through the induction of senescence in the MCF-7 human breast cancer cell line. The expression of IGFBP-3 resulted in morphological changes shown in senescent cells, such as a flattened cytoplasm and increased granularity, as well as a marked increase in the percentage of cells containing SA-ß-galactosidase. Furthermore, BrdUrd-Hoechst flow cytometry showed that the expression of IGFBP-3 increased the fraction of non-cycling cells that had progressed through three cell cycles, indicating IGFBP-3 induces senescence. Previous studies have reported that IGFBP-3 inhibits cell proliferation through the induction of apoptosis^13,14. In the present study, we used a pan-caspase inhibitor (zVAD-fmk) to demonstrate that the inhibitory effect of IGFBP-3 on cell growth may be caused by the mechanisms other than caspase-mediated apoptosis. Treatment with a pan-caspase inhibitor (zVAD-fmk) did not totally abolish the growth-inhibitory effect of IGFBP-3, while it abolished the growth-inhibitory effect of TNF-α. In addition, the pan-caspase inhibitor did not reverse IGFBP-3-induced morphological changes indicative of senescence, and it did not reduce IGFBP-3-induced SA-ß-galactosidase activity. These results suggest that IGFBP-3 inhibits cell proliferation in part by the induction of senescence, in addition to the induction of caspase-mediated apoptosis in MCF-7 human breast cancer cells, as reported in previous studies^13,14.

Cellular senescence is defined as a state of stable cell cycle arrest in response to diverse signaling. It is known that three contexts for arrest of the cell division cycle, quiescence, differentiation, and senescence, exist in multicellular organisms to maintain their homeostasis³⁹. Quiescence is a reversible growth arrest state manipulated by growth stimuli or cell density, whereas terminal differentiation and senescence are permanent post-mitotic states in which cells remain metabolically active while not dividing. Senescent cells show no response to mitogens and are permanently arrested in the G0/G1 phase in the cell cycle. Senescence is observed in normal somatic cells, which ceases proliferation after a defined number of cell divisions. This permanent growth arrest, resulting from telomeric shortening at each cell division, is referred to as replicative senescence^27,28. In addition, a senescence-like state, called stress-induced premature senescence or accelerated senescence, can be observed in cells irrespective of telomere length when they are exposed to oncogenic mutations, oxidative stress, inadequate culture conditions, or DNA damaging agents^40,41. Therefore, senescence is thought to function as an additional barrier against an accumulation of damaged DNA, resulting in the transformation of cells, as well as an underlying cause of aging²⁷. Stress-induced premature senescence also shows similar morphological and biochemical features to those observed in replicative senescence, suggesting that senescence is a common response to cellular damage, such as DNA damage. IGFBP-3 may be involved in both replicative senescence and stress-induced premature senescence. Previous studies have suggested that the expression of IGFBP-3 is increased with age in human dermal fibroblasts, epithelial cells, and umbilical vein endothelial cells^22–24, as well as in breast cancer cells treated with chemotherapeutic drugs²⁶ and premature senescent fibroblasts exposed to tert-butylhydroperoxide and ethanol⁴².

Many studies have elucidated the underlying molecular mechanisms and diverse pathways of senescence. Effector mechanisms of senescence, such as a senescence-associated secretory phenotype, have been identified^43,44. However, the alterations of gene expression and collective effector phenotypes in senescent cells can vary depending on cell types and triggering events. Moreover, it has been reported that the gene expression profiles for senescence activated by telomeric shortening only partially overlaps with senescence induced by various stressful stimuli⁴⁰. Although senescence has a complex and diverse nature, general evidence suggests that p53 and/or pRb protein along with their regulators, such as p21, p16, and ADP ribosylation factor (ARF), are involved in the progression of senescence^39–41. In our previous study, we demonstrated that IGFBP-3 induced G1 cell cycle arrest by the inhibition of cyclin D1, cyclin D3, cyclin E, cyclin A, CDK2, CDK4, and total and phospho-pRb, as well as by the increase of p21 and p16 in MCF-7 breast cancer cells¹⁷. This study suggests that pRb pathways, at least in part, may be involved in IGFBP-3-induced senescence, as the G1 cell cycle arrest includes both quiescence and senescence responses. The exact molecular mechanism of senescence by IGFBP-3 remains to be established.

Senescence is linked to telomere length and telomere dysfunction. Telomere shortening with each replication cycle results from the inability of DNA polymerase, telomere dysfunction by either a change of telomeric DNA structure or sequence, or depleted and mutated telomere proteins, leading to permanent growth arrest. These mechanisms may reduce the risk of tumor formation⁴⁰. Telomerase, a ribonucleoprotein complex with reverse transcriptase activity, can catalyze the synthesis of telomeric DNA onto the ends of chromosomes and provide unlimited proliferative capacity to the cells for bypassing senescence^27,28. Although telomerase activity is undetectable in most normal adult cells, it can be detected in fetal tissue, bone marrow stem cells, testes, peripheral blood lymphocytes, epidermis, and intestinal crypt cells³⁰. In contrast, the vast majority of human cancer cells have a relatively high level of telomerase activity, which immortalizes the cells. In the present study, we aimed to determine whether IGFBP-3 modulated telomerase activity in the process of inducing senescence in MCF-7 human breast cancer cells. We demonstrated that the expression of IGFBP-3 decreased telomerase activity using TRAP assays. The present study further demonstrated that IGFBP-3 inhibited telomerase activity by modulating the expression of telomerase components, such as hTR and hTERT, at the mRNA and protein levels. In addition, we demonstrated that exogenous IGFBP-3 decreases hTERT mRNA and protein expressions, and telomerase activity in hTERT-overexpressed normal breast cell. These results suggest that the inhibition of telomerase activity by IGFBP-3 is a potential mechanism for IGFBP-3-induced senescence in MCF-7 cells. However, the mechanism underlying the potential inhibition of telomerase by IGFBP-3 remains elucidated. In addition, post-translational modifications (e.g., phosphorylation, glycosylation, and acetylation) of IGFBP-3 may influence protein activity, interaction and localization, thereby functioning as a key regulator for IGFBP-3 action⁴⁵. The occurrence and identification of post-translational modifications were not considered in this study; therefore, biological roles of post-translational modifications on IGFBP-3 should be elucidated in further studies.

This study demonstrated that IGFBP-3 induced senescence by inhibiting telomerase activity, thereby inhibiting cell proliferation in MCF-7 human breast cancer cells. In previous studies, IGFBP-3 has been reported to inhibit cell proliferation through the induction of apoptosis or cell cycle arrest. The demonstration of the involvement of IGFBP-3 in the senescence of breast cancer cells in this study presents evidence of another potential mechanism for the anti-proliferative effect of IGFBP-3, as well as its potential therapeutic modality for cancer treatment.

4.1. Reagents and Antibodies

Dulbecco’s modified Eagle medium (DMEM) and trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA) were purchased from Invitrogen (Carlsbad, CA, USA). DMEM:F-12 media was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Tissue culture reagents and plastics were obtained from Mediatech (Herndon, VA, USA), Becton Dickinson (Franklin Lakes, NJ, USA), Nunc (Naperville, IL, USA), and Sigma-Aldrich (Saint Louis, MO, USA). Ecdysone-inducible expression system was purchased from Invitrogen (Carlsbad, CA, USA). CellTiter 96 cell proliferation assay was purchased from Promega (Madison, WI, USA), and z-VAD-fluoromethylketone (fmk) (broad-spectrum caspase inhibitor) from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). Human IGFBP-3 antibody was purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase-labeled IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IGFBP-3 recombinant protein was purchased from Eagle Biosciences (Amherst, NH, USA).

4.2. Cell Culture

The MCF-7 human breast cancer cell line was obtained from American Type Culture Collection (Rockville, MA, USA). The MCF-7 cell, which is an estrogen receptor positive cell line, shows little-to-no secretion of IGFBP-3 under basal condition^33,34. The cells were maintained in DMEM supplemented with 4.5 g/L glucose, 110 mg/L sodium pyruvate, and 10% fetal bovine serum (FBS). The cells were cultured at 37°C in 5% CO₂ and 100% humidity. At 60–70% confluence, the cells were washed with fresh serum-free media (SFM) and then incubated in phenol red-free SFM in the presence of 15 µM ponasterone A for an additional 72 hours to express IGFBP-3.

4.3. Establishment of MCF-7-derived Cell Line Inducibly Expressing IGFBP-3

An MCF-7-derived cell line inducibly expressing IGFBP-3 was established as previously described¹⁴. A cDNA fragment containing a 1.5-kb human IGFBP-3 (equivalent to nucleotides 674–2191 of the cDNA sequence) was ligated into the pIND expression vector, which contained five modified ecdysone response elements. This construct was co-transfected with a second plasmid (pVgRXR), expressing the ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), into MCF-7 cells using the FuGENE 6 transfection reagent (Roche, Indianapolis, IN). After 48 hours, the cells were split into selective medium containing G418 (800 µg/mL) for pIND/IGFBP-3 and zeocin (100 µg/mL) for pVgRXR, and the foci were picked to generate a stable cell line expressing pVgRXR and pIND/IGFBP-3. Ponasterone A, which is an ecdysone analog inducing heterodimers of RXR and VgEcR to bind the hybrid ecdysone response element in the pIND vector, was used to express IGFBP-3 in an inducible manner. MCF-7 cells transfected with pVgRXR were used as a negative control (EcR cells).

4.4. GGG-IGFBP-3 Mutant cDNA Generation and IGFBP-3 Plasmids Transfections in MCF-7 Cells

The GGG-IGFBP-3 mutant cDNA was generated by site-directed mutagenesis at residues Ile56, Leu80, and Leu81 to Gly56, Gly80, and Gly81, as described previously³⁵. Binding studies (including BIAcore analysis) showed that the GGG-IGFBP-3 mutant protein, generated in E. coli and baculovirus expression systems, abolished the affinity for IGFs.³⁵ For transfection, GGG-IGFBP-3 mutant cDNAs were subcloned into the pCMV6 vector as previously described. MCF-7 cells were seeded in DMEM supplemented with 10% FBS and were transfected with pCMV6 vector or pCMV6/GGG-IGFBP-3 using the FuGENE 6 transfection reagent. After incubation for 5 hours, the transfected cells were washed by phosphate-buffered saline (PBS) and replaced with fresh serum-free media. Samples for real-time PCR were obtained 48 hours after transfection.

4.5. hTERT Plasmid Transfection in Human Normal Breast Cells

Human normal breast MCF-12F cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MCF-12F cells were cultured in DMEM:F-12 media with 20 ng/mL epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL human insulin, 500 ng/mL hydrocortisone, and 5% chelex-treated horse serum. Cells were incubated at 37°C in a humidified incubator with 5% CO2. Lipofectamine 2000 transfection reagent and hTERT plasmid DNA were purchased from Invitrogen (Carlsbad, CA, USA) and OriGene (Rockville, MD, USA), respectively. Cells were seeded in 6-well plates at the concentration of 2 x 10⁵ cells/well. At 70 ~ 80% confluence, 500 ng hTERT plasmid DNA were transfected into MCF-12F cells with antibiotic-free Opti-MEM using Lipofectamine 2000 according to the manufacturer’s instructions. After incubation for 5 hours, the transfected cells were washed by PBS and replaced with fresh serum-free media with designated concentrations of the exogenous IGFBP-3. The samples for immunoblotting and real-time PCR were obtained 48 hours after medium change.

4.6. Small Interfering RNA Transfection

Small interfering RNA (siRNA) pool against human IGFBP-3 (siRNA:IGFBP-3, siGENOME SMARTpool M-004777-01-0005) and mismatch control (ON-TARGETplus siCONTROL, D-001810-01-05) were purchased from Dharmacon Research (Lafayette, CO, USA). The SMARTpool siRNA against IGFBP-3 is composed of 4 different siRNAs against 4 different target sequences in the IGFBP-3, but not individual siRNA. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect 50 nM siRNA:IGFBP-3 into the cells. Transfected cells were then incubated with 10% FBS in growth media for 24 hours.

4.7. Cell Proliferation Assay

Assays were performed at the time indicated in the text. Culture media was aspirated, followed by adding 100 µl of CellTiter 96 Aqueous one solution (1:5 dilution with phenol red-free SFM) directly to culture wells and incubating for 1 hour at 37°C. The quantity of viable cells was measured in a 96-well plate reader at 490 nm. We used 50 nM siRNA:IGFBP-3 to suppress the expression of IGFBP-3 or 25 µM Z-VAD-fmk to suppress the overall caspase activity, which was shown to completely inhibit apoptosis in the same cell line in our previous study¹⁴.

4.8. Flow Cytometric Analysis of Non-cycling Cells

A modified BrdUrd-Hoechst quenching technique to quantify the fraction of non-cycling cells was performed according to a previously described method³⁶. Cells were grown in 6-well plates until 60–70% confluent, and then the media were replaced with SFM in the presence or absence of 15 µM ponasterone A or 100 mM BrdUrd for 24 hours. Both attached and floating cells were harvested by trypsinization, pelleted at 1000 rpm for 5 minutes, and washed three times with PBS. Each cell sample was stored in DMEM containing 10% DMSO at -20°C until analyzed. Cells were thawed and stained with 2 µg/mL Hoechst 33258 (Sigma) for 10 minutes at 4°C. Cells were analyzed for cell number versus Hoechst fluorescence using an LSR-II flow cytometer (Becton Dickinson, Franklin Lakes, NJ USA) equipped with two argon lasers. The first laser was tuned to 488 nm (15 mW output), and the second was tuned to 360 nm (10 mW output). The mean percentage of the non-cycling cells was determined. Three independent experiments were completed for this analysis.

4.9. Senescence-associated β-galactosidase Activity

Senescence-associated ß-galactosidase (SA-ß-galactosidase) staining was performed according to a previously described method³⁷. Sub-confluent cells were washed twice in PBS, fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS, and rinsed twice with PBS. The cells were stained with a staining solution (0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactoside, 40 mM citric acid, sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂). Then, the cells were incubated for 6 hours at 37°C. The cells were rinsed twice with PBS after incubation. Approximately 1000 cells in each plate were counted at 200X magnification. The proportion of cells exhibiting a medium- to dark-blue stain, indicative of SA-ß-galactosidase activity, was then scored. Duplicate plates/cell lines were used in each experiment. Three independent experiments were completed.

4.10. Preparation of Cell Lysates

Cell lysates were prepared as described previously with minor modifications²⁵. In brief, confluent cells were washed with cold PBS and then scraped from plates in the presence of cold lysis buffer containing 20 mM Tris (pH 8.0), 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 0.5% sodium deoxycholate, and Complete Protease Inhibitor mixture (Boehringer Mannheim). Floating cells in the conditioned media were collected by centrifugation and resuspended in cold lysis buffer. Cell lysates obtained from confluent cells and floating cells were centrifuged for 15 minutes at 4°C to remove cell debris. The aliquots were stored at -70°C until further use.

4.11. Western Blotting Analysis

The cell lysates were electrophoresed on a 12% SDS-polyacrylamide gel under reducing conditions and electro-transferred onto polyvinylidene fluoride membranes. A standard blotting protocol was performed using anti-human IGFBP-3 antibodies (1:500 dilution) followed by peroxidase-conjugated IgG (1:3,000 dilution). Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK).

4.12. Telomeric Repeat Amplification Protocol Assay

Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) using the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche, Penzberg, Germany) in accordance with the manufacturer’s instructions. Briefly, 2 × 10⁶ cells were lysed in 200 µL of ice-cold lysis buffer for 30 minutes. Then, the lysate was centrifuged at 16,000 × g for 20 minutes at 4°C, and the supernatants were collected. Control templates were included in the kit, which contained positive-telomerase template DNA with the same sequence as a telomerase product with eight telomeric repeats. Two microliters of each extract (corresponding to 2 × 10³ cell equivalents) were assayed for telomerase activity in a 50 µL reaction using a modified TRAP assay. Telomeric repeats were added to a biotin-labeled primer during the first telomerase-mediated extension reaction. Then, the elongation products were amplified in a PTC-220 DNA Engine Dyad PCR machine (MJ Research, Waltham, MA). An aliquot of the PCR product was denatured, hybridized to a DIG-labeled, telomeric repeat-specific probe, and bound to a streptavidin-coated 96-well plate. Finally, the immobilized PCR product was detected with an anti-DIG-horseradish peroxidase coupled antibody, visualized by a color reaction product using the substrate tetramethylbenzidine, and semi-quantified photometrically at 450 nm using a FLUORstar Optima microplate reader (BMG Labtechnologies, Germany). Negative controls were prepared from each cell extract by heat-inactivation of the telomerase for 10 minutes at 85°C

4.13. Reverse-transcription Quantitative PCR

Total RNA was isolated from the cells using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Five microgram of total RNA was subjected to reverse transcription using Superscript TM III first-strand synthesis system (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed in 20 µl of reaction mixtures containing cDNA, TaqMan primer pairs, and TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA, USA). The primer pairs were obtained from Applied Biosystems (Hs00972650 for hTERT, TR-ANY for hTR, Hs99999903 for β-actin). Amplification was performed in duplicate with the ABI 7300 system (Applied Biosystems) with the following profile: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Gene expression for each sample was expressed as the threshold cycle (Ct) normalized to β-actin (ΔCt). The ΔCt values were compared between samples from ponasterone A-treated cells and control samples (cells in serum-free media) to calculate ΔΔCt. The final comparison of transcript ratios between samples was given as 2^ΔΔCt30.

4.14. Densitometric and Statistical Analysis

Densitometric measurements of immunoblots were performed using a Bio-Rad GS-670 Imaging densitometer (Bio-Rad, Melville, NY, USA). Statistical comparisons among groups were performed using a non-parametric Mann-Whitney U test (IBM SPSSA statistics ver. 25). We considered a P value of 0.05 or less as significantly different.

Acknowledgments: Not applicable.

Author Contributions: Conceptualization, H-S.K. and Y.O.; methodology, A.K. and W.J.L.; validation, A.K. and H.W.C.; formal analysis, A.K. and H-S.K.; investigation, A.K., W.J.L., J.H.K, Y.J.K., J.A.; resources, Y.O.; data curation, A.K. and W.J.L.; writing—original draft preparation, A.K.; writing—review and editing, H-S.K.; visualization, A.K.; supervision, Y.O.; project administration, H-S.K.; funding acquisition, H-S.K. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Competing Interests Statement: The authors have no potential conflicts of interest to disclose.

Funding: This research was funded by the Novo Nordisk, Grant number 7-2009-0572.

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No competing interests reported.

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Insulin-Like Growth Factor Binding Protein-3 Induces Senescence by Inhibiting Telomerase Activity in MCF-7 Breast Cancer Cells

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Abstract

Figures

1. Introduction

2. Results

2.1. IGFBP-3 Inhibits Proliferation of MCF-7 Cells

2.2. The Inhibitory Effect of IGFBP-3 Is Mediated by a Mechanism Other Than Apoptosis

2.3. IGFBP-3 Increases the Fraction of Non-cycling Cells

2.4. IGFBP-3 Induces Senescence-like Phenotype

2.5. Senescence-associated ß-galactosidase Activity Is Increased by IGFBP-3

2.6. IGFBP-3 Decreases Telomerase Activity

2.7. IGFBP-3 Decreases Reverse Transcriptase Components and RNA of Telomerase

3. Discussion

4. Materials And Methods

4.1. Reagents and Antibodies

4.2. Cell Culture

4.3. Establishment of MCF-7-derived Cell Line Inducibly Expressing IGFBP-3

4.4. GGG-IGFBP-3 Mutant cDNA Generation and IGFBP-3 Plasmids Transfections in MCF-7 Cells

4.5. hTERT Plasmid Transfection in Human Normal Breast Cells

4.6. Small Interfering RNA Transfection

4.7. Cell Proliferation Assay

4.8. Flow Cytometric Analysis of Non-cycling Cells

4.9. Senescence-associated β-galactosidase Activity

4.10. Preparation of Cell Lysates

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References

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