Abstract
Background/Aim: The antitumor effect of sustained calcium supply on Src degradation was investigated in the context of hormone-dependent breast cancer, followed by elucidation of the underlying mechanisms. Materials and Methods: Hormone-dependent T-47D breast cancer cells were used. Lactate calcium salt (LCS) was used as the source of sustained calcium supply, and the applicable concentration of LCS was determined by the colorimetric MTT assay. LCS-mediated deactivation of downstream signaling via Src degradation was identified by western blot and immunocytochemistry. Results: Calcium-mediated degradation of Src decreased survival signaling via phosphoinositide 3-kinase and protein kinase B and resulted in significant inhibition of the clonogenic ability of hormone-dependent breast cancer cells. Tumor volume was significantly decreased in response to LCS injection in a heterotopic xenograft model, and immuno histochemistry revealed tumor necrosis. Conclusion: Sustained supply of calcium inhibited survival signaling via degradation of Src in hormone-dependent breast cancer cells.
Breast cancer is characterized by the expression of certain biomarkers. Estrogen receptor (ER) and progesterone receptor (PR) are used as biomarkers for targeting hormone-dependent breast cancer (1). Most patients with breast cancer require systemic therapy prior to surgery to improve prognosis (2). Systemic therapy can also be provided post-surgery if the treatment outcomes or biomarkers indicate an increased risk of recurrence (3). Most patients with hormone receptor-positive breast cancer, independent of the human epidermal growth factor receptor (EGFR) status, are provided with the option of endocrine therapy to inhibit the activity of hormone receptors (4). It has been suggested that even when the expression of EGFR is low or undetectable, the dynamic changes in the tumor microenvironment greatly reduce the response to therapeutics (5). A representative case is the internalization of the ER-estrogen complex that is transported into the cytoplasm and nucleus to promote breast cancer cell survival via uncontrolled transcriptional activation despite the presence of appropriate ER blockade (6). Therefore, it is essential to develop a method for enhancing the efficacy of the existing endocrine therapy. Src is involved in ligand-dependent activation of ER receptor signaling, thereby contributing to the activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling (7). PI3K/AKT signaling plays an important role in the survival and proliferation of hormone-dependent breast cancer cells (Figure 1). Our previous studies have shown the anti-cancer effects of continuous calcium treatment in metastatic colorectal cancer cells, and that they are mediated via the suppression of survival signaling through the proteasomal degradation of Src and focal adhesion kinase (8). Therefore, we anticipated that calcium-dependent Src degradation can have a potential anticancer effect in the context of hormone-dependent breast cancer.
In this study, we investigated the possibility of suppressing various Src-mediated survival signals of cancer cells by a continuous calcium supply. In addition, the anticancer effect of lactate calcium salt (LCS) was confirmed in the context of hormone-dependent breast cancer cells.
Materials and Methods
Cell culture and reagents. The hormone-dependent human breast cancer cell line (T-47D) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). T-47D cells were cultured in RPMI-1640 medium (Welgene, Daegu, Republic of Korea), supplemented with 10% fetal bovine serum (Welgene) and 1% penicillin/streptomycin (Welgene) at 37°C in an atmosphere containing 5% CO2. LCS and calpeptin were purchased from Sigma (St Louis, MO, USA).
Estrogen-dependent signaling pathway in the context of breast cancer survival. Estrogen receptor (ER) is activated upon binding to estrogen (E2), and subsequently interacts with phosphoinositide 3-kinase (PI3K) and Src to activate the serine/threonine-protein kinase, AKT. The ER pathway plays an important role in the proliferation and survival of breast cancer cells.
Measurement of T-47D cell viability following treatment with different concentrations of lactate calcium salt (LCS). A: Photograph showing the difference in viability of breast cancer cells with respect to the treatment time and LCS concentration. B: Representative microphotographs of apoptotic morphology T-47D cells following treatment with 1, 2.5, and 5 mM LCS. C: Quantitative analysis graph depicting percent viability of T-47D cells. The experiments were performed in quintuplicate. Significantly different at: *p<0.05 and **p<0.001 vs. control group; #p<0.001 vs. the control, 1, and 2.5 mM groups. Results are displayed as the mean±SD.
Determination of the effect of sustained calcium supply on the expression of Src, phosphoinositide 3-kinase (PI3K), and protein kinase B (AKT). A: Confocal images showing immunocytochemical staining for Src in T-47D cells following lactate calcium salt (LCS) treatment. Scale bar, 10 μm. B: Western blot for Src expression and quantitative analysis in T-47D cells following LCS treatment. C: Confocal images showing immunocytochemical staining of PI3K in T-47D cells following LCS treatment. Scale bar, 10 μm. D: Western blot for PI3K expression and quantitative analysis in T-47D cells following LCS treatment. E: Confocal images showing AKT immunocytochemical staining in T-47D cells following LCS treatment. Scale bar, 10 μm. F: Western blot for AKT expression and quantitative analysis in T-47D cells following LCS treatment. The experiments were performed in quintuplicate. Significantly different at **p<0.001 vs. the control group. The results are displayed as the mean±SD.
Cell viability assay. T-47D cells were cultured in a 96-well plate (3×103 cells/well) for 24 h, and then treated with different concentrations of LCS (1, 2.5, and 5.0 mM) for 24, 48, or 72 h at 37°C. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added to each well, and the cells were incubated at 37°C for 1 h in a humidified environment containing 5% CO2. After the media was discarded, 200 μl of dimethyl sulfoxide (Cell Signaling Technology, Danvers, MA, USA) were added to each well. The absorbance was read at 570 nm using a microplate reader (iMark Microplate Absorbance Reader, Bio-Rad, CA, USA).
Correlation between the activation of the calcium-dependent proteolytic enzyme calpain and inhibition of Src and subsequent downstream signaling in T-47D cells. A: Immunocytochemistry for determining the effect of calcium-mediated calpain activity on Src expression in T-47D cells. Scale bar, 10 μm. B: Western blot and quantitative analysis for evaluating the effect of calcium-mediated calpain activity on Src expression in T-47D cells. C: Immunocytochemistry for determining the effect of calcium-mediated calpain activity on phosphoinositide 3-kinase (PI3K) expression in T-47D cells. Scale bar, 10 μm. D: Western blot and quantitative analysis for evaluating the effect of calcium-mediated calpain activity on PI3K expression in T-47D cells. E: Immunocytochemistry for determining the effect of calcium-mediated calpain activity on protein kinase B (AKT) expression in T-47D cells. Scale bar, 10 μm. F: Western blot and quantitative analysis for evaluating the effect of calcium-mediated calpain activity on AKT expression in T-47D cells. The experiments were performed in quintuplicate. Significantly different at: **p<0.001 vs. the control. The results are displayed as the mean±SD.
Immunocytochemistry. T-47D cells were seeded onto a bio-coated coverslip (BD Bioscience, San Jose, CA, USA) at a density of 1×105 cells for 24 h. Then, the cells were treated with LCS alone or in combination with calpeptin for 6 h and fixed with 4% paraformaldehyde after washing with PBS. The cells were incubated for 15 h at 4°C with the following primary antibodies: Src (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA); PI3K (1:300 dilution, Santa Cruz Biotechnology); AKT (1:300 dilution, Santa Cruz Biotechnology). After a PBS wash, the cells were incubated with a biotinylated anti-rabbit secondary antibody (1:2000 dilution, Vector Laboratories, Burlingame, CA, USA) and fluorescein-conjugated streptavidin (Vector Laboratories) for visualization. The cover-slips were mounted on slides using VECTASHIELD® Hard Set™ mounting medium containing 4’,6-diamidino-2-phenylindole (Vector Laboratories). Images were obtained by confocal laser scanning microscopy (LSCM, Nikon A1+, Tokyo, Japan).
Protein extraction. T-47D cells were seeded in a 6-well plate at a density of 1×106 cells/well. After incubation, the cells were treated with LCS alone or in combination with calpeptin, incubated for 6 h at 37°C, harvested, and washed once with ice-cold PBS. The washed cells were lysed in RIPA lysis buffer (Roche, Basel, Switzerland) and centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was used for western blot.
Western blot analysis. Proteins were separated (20 μg protein per lane) on 8% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Then, the membranes were blocked in 5% non-fat milk (Bio-Rad) for 1 h at 25°C and incubated overnight at 4°C with primary antibodies diluted in Tris-buffered saline containing Tween 20 (TBST), 5% bovine serum albumin, and 0.1% sodium azide (Sigma-Aldrich). The following specific primary antibodies were used: anti-actin (1:1000 dilution, Santa Cruz Biotechnology); anti-Src (1:1000 dilution, Cell Signaling, Danvers, MA, USA); anti-PI3K (1:1000 dilution, Santa Cruz Biotechnology); anti-AKT (1:1000, Santa Cruz Biotechnology). The membranes were washed with TBST and incubated with secondary antibodies at 25°C for 2 h. The following secondary antibodies were used: anti-rabbit secondary antibody (1:10,000 dilution, Abclone, Seoul, Republic of Korea); anti-mouse secondary antibody (1:10,000 dilution, Abclone). Immunoblots were developed using a western blot detection reagent (Abclone) and were exposed to x-ray film (Agfa, Leverkusen, Germany) according to the manufacturer's protocol.
Clonogenic assay. T-47D cells were seeded at a density of 3×102 cells/well in a 6-well plate and incubated for 24 h at 37°C. After 24 h, the media were removed, and the cells were treated with LCS for 14 days in a humidified atmosphere containing 5% CO2 at 37°C. Next, the colonies were fixed with methanol, stained with hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA), and counted under an optical microscope (Olympus, Center Valley, PA, USA).
Xenograft animal model. All experiments were performed under the institutional guidelines established by the Institutional Animal Care and Use Committee of Gachon University, Incheon, Republic of Korea (IACUC-LCDI-2019-0102). Five-week-old BALB/c nude mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All animals were maintained in a 12 h light/dark cycle (light on, 08:00 h) at 22 to 25°C with ad libitum access to food and water. T-47D cells (1×107) were injected into the hind flank of mice. Seven mice were assigned to the control group, and 10 mice were assigned to the LCS-treated group. When the tumor grew to about 150-200 mm3, 20 mg/kg of LCS was subcutaneously injected for 21 days. Tumor size was measured three times per week using a digital caliper, and the tumor volume was calculated using the following formula: V=(L×W2)/2 (L, length; W, width). At the end of the experiment, all tumors were harvested for histological analysis.
Statistical analysis. All data are presented as the mean±standard deviation (SD). Statistical significance was analyzed using the Student's t-test and one-way ANOVA depending on whether the data was normalized. A difference of p<0.05 was considered to be statistically significant (Sigmastat ver. 3.5, Systat Software Inc., Chicago, IL, USA).
Results
Sustained calcium supply gradually decreased the viability of T-47D cells. Cell viability was evaluated as a function of increasing LCS concentration (1, 2.5, and 5 mM) to select the optimal dose for experiments in T-47D cells. A microphotograph of T-47D cells in the 96-well plate is shown in Figure 2A; cell death was clearly observed following treatment with increasing LCS concentrations. Quantitative analysis revealed no significant change in cell viability following treatment with 1 mM LCS for up to 72 h relative to the control (Figure 2B). The percent survival was significantly decreased to 68.48±8.93% and 64.26±10% after 48 and 72 h, respectively, upon treatment with 2.5 mM LCS, and to 82.04±4.69%, 47.38±2.39%, and 30.41±2.35% after 24, 48, and 72 h, respectively, upon treatment with 5 mM LCS relative to control (Figure 2B).
Sustained calcium supply induced Src degradation and inhibited PI3K/AKT signaling. The effect of sustained calcium supply on the expression of Src, PI3K, and AKT was investigated in T-47D cells (Figure 3). Figures 3A, C, and E show a decrease in the expression of Src, PI3K, and AKT, respectively, as evaluated by immunocytochemistry. The expression of Src, PI3K, and AKT in whole-cell extracts decreased following LCS treatment (Figures 3B, D and F). Quantitative analysis revealed significant reduction in Src, PI3K, and AKT expression in LCS-treated cells compared to the control (Figures 3B, D and F).
Sustained calcium supply activated the proteolytic enzyme calpain to mediate Src degradation and inhibition of downstream signaling in T-47D cells. Next, we investigated protein degradation via the activation of the calcium-dependent proteolytic enzyme calpain. The expression of Src, PI3K, and AKT was verified following treatment with calpeptin, a calpain inhibitor (Figure 4). Immunocytochemistry showed that a single treatment with LCS reduced the expression of Src, PI3K, and AKT in T-47D cells, and the LCS-induced reduction in the expression of Src, PI3K, and AKT was restored following treatment with calpeptin (Figures 4A, C and E). Quantitation of the results is indicated in Figures 4B, D and F.
Sustained calcium supply reduced the proliferation of hormone-dependent breast cancer cells. To investigate the in vitro anticancer effect of sustained calcium supply in hormone-dependent breast cancer cells, the clonogenic ability of the cells was examined following LCS treatment. The representative images of colony formation assay following sustained calcium supply are presented in Figure 5A (a single colony has been magnified to reveal the morphological features). The single colony was compact and smaller than those in the control after sustained calcium supply (Figures 5A). Quantitative analysis indicated that the number of colonies was significantly reduced to 169.3±25.67 compared to control (349.67±27.52; Figure 5B).
Sustained calcium supply slowed down the growth of hormone-dependent breast cancer. A schematic illustration of the experimental design is displayed in Figure 6A. To confirm the antitumor effect of sustained calcium supply in vivo, tumor volume was measured three times per week following LCS treatment (Figure 6B). In the LCS-treated group, the tumor volume was significantly decreased after 12 days (fifth measurement in Figure 6B) compared to that in the control group (Figure 6B). On the last day of tumor measurement, the tumor volume was 314.34±135.89 mm3 (average) in the LCS-treated group and 1440.39±273.7 mm3 (average) in the control group (Figure 6B). The representative images comparing tumor growth in the control and LCS-treated group are shown in Figure 6C. Tumor growth was reduced in the LCS-treated group compared to that in the control group (Figure 6C). Following LCS treatment, cell necrosis was observed in the tumor mass, even at the periphery of the tumor. The tumors in LCS-treated mice exhibited a collapsed internal structure, with almost no nuclear staining (Figure 6D).
Discussion
In this study, we investigated whether sustained calcium supply could result in anti-tumor effects in hormone-dependent breast cancer cells. Calcium-mediated Src degradation resulted in inhibition of the expression of the downstream signaling molecules, PI3K and AKT. These effects reduced the proliferation of hormone-dependent breast cancer cells and inhibited tumor growth in an animal xenograft model.
The source of calcium for all in vitro experiments in this study was 5 mM LCS. The rationale behind selecting 5 mM as the concentration of LCS was the low viability of breast cancer cells at this concentration, regardless of treatment time. In our previous studies targeting colorectal cancer, we mostly used LCS at concentrations that could reduce cancer cell viability from 100% to about 60% in vitro, as in these studies LCS was used in combination with conventional anticancer agents (8). Our current study, to our knowledge, is the first attempt to investigate the effectiveness of sustained calcium supply using LCS. Therefore, the optimal LCS concentration was selected to reduce cancer cell viability to about 40% and to elucidate the mechanism underlying the antitumor effect of LCS. Additionally, 5 mM LCS release about 1 mM calcium, which does not exceed the physiological concentrations of calcium.
The mechanism presented in Figure 1 indicates that hormone-dependent breast cancer is dependent on Src-mediated signaling along with ER activation. As Src-mediated phosphorylation of mediators of signaling cascades is necessary for cancer-cell proliferation, and because Src regulates many signaling pathways involved in the development of cancer (7, 9), we anticipated that anti-cancer effects could be investigated through the inhibition of intracellular signal transduction. The complex crosstalk between Src and PI3K is involved in the regulation of cancer cell proliferation. Further, AKT, which is part of the Src and PI3K signaling cascade, is involved in tumor metabolism, growth, proliferation, and survival (10). Our results demonstrated that sustained calcium supply resulted in the reduction in the expression of PI3K and AKT, along with Src destabilization. We verified that the anticancer effect associated with sustained calcium supply was also observed in vivo, as evidenced by inhibition of survival and colonization of breast cancer cells.
Confirmation of the reduced clonogenic ability of hormone-dependent breast cancer cells in response to sustained calcium supply. A: Representative images of T-47D colonies following treatment with LCS. B: Quantitative analysis of the number of T-47D colonies. The experiments were performed in quintuplicate. Significantly different at: **p<0.001 vs. the control group. The results are displayed as the mean±SD.
Confirmation of the antitumor effect of sustained calcium supply on hormone-dependent breast cancer in vivo. A: A Schematic diagram of the in vivo experiment. B: Comparison of the tumor growth between control and LCS-treated groups. C: Representative images of tumor mass. D: Hematoxylin and eosin (H&E) staining for comparing tumor necrosis between the control and LCS-treated groups. Scale bar, 100 μm. Significantly different at: **p<0.001 vs. the control group. The results are displayed as the mean±SD.
Calpain is a protease that is activated by calcium (11). Recently, it has been implicated in regulating the cleavage of many adhesion-associated and actin-regulatory proteins (12). The role of calpain in regulating the expression of Src has not been elucidated. In this study, the role of calcium-dependent calpain was investigated using the calpain inhibitor, calpeptin. Treatment with calpeptin resulted in the recovery of PI3K and AKT, as well as of Src expression. Thus, the decrease in Src levels was indeed a result of calcium-mediated activation of calpain.
It has been shown that after LCS wash out, the intracellular concentration of calcium was rapidly depleted within 480 sec; this could due to cellular permeation (13). Therefore, daily treatment was required for sustained calcium supply. Further research is required to determine the effective dose of LCS for targeting hormone-dependent breast cancer. In addition, investigation of the optimal administration route to ensure delivery to the exact site of the origin of cancer, without the interference of first-pass effects should be considered.
Taken together, sustained calcium supply-induced Src proteolysis resulted in a significant decrease in PI3K and ATK expression and inhibition of hormone-dependent breast cancer cell proliferation. Therefore, a steady calcium supply could help in the management of hormone-dependent breast cancer.
Acknowledgements
This work was supported by the Gachon University, Incheon, Republic of Korea [grant number 2019-0327].
Footnotes
* These Authors contributed equally to this study.
Authors' Contributions
Keun-Yeong Jeong and Sun Young Park designed the experiments. Sun Young Park, Jae Ki Lee, and Min Hee Park performed the experiments. Keun-Yeong Jeong, Hwan Mook Kim, Sun Young Park, and Jae Ki Lee analyzed the data. Keun-Yeong Jeong and Sun Young Park wrote the paper.
Conflicts of Interest
There are no potential conflicts of interest to declare with respect to this study.
- Received March 2, 2020.
- Revision received March 15, 2020.
- Accepted March 16, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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