Suppressing Src-Mediated EGFR Signaling by Sustained Calcium Supply Targeting Triple-Negative Breast Cancer.

Src is emerging as a promising target in triple-negative breast cancer (TNBC) treatment because it activates survival signaling linked to the epidermal growth factor receptor. In this study, the effect of calcium supply on Src degradation was investigated to confirm underlying mechanisms and anticancer effects targeting TNBC. MDA-MB-231 cells, the TNBC cell line, were used. Calcium supply was feasible through lactate calcium salt (CaLac), and the applicable calcium concentration was decided by changes in the viability with different doses of CaLac. Expression of signaling molecules mediated by calcium-dependent Src degradation was observed by Western blot analysis and immunocytochemistry, and the recovery of the signaling molecules was confirmed following calpeptin treatment. The anticancer effect was investigated in the xenograft animal model. Significant suppression of Src was induced by calcium supply, followed by a successive decrease in the expression of epithelial growth factor receptor, RAS, extracellular signal-regulated kinase, and nuclear factor kappa B. Then, the suppression of cyclooxygenase-2 contributed to a significant deactivation of the prostaglandin E2 receptors. These results suggest that calcium supply has the potential to reduce the risk of TNBC. However, as this study is at an early stage to determine clinical applicability, close consideration is needed.

Remodeling of Cancer-Specific Metabolism under Hypoxia with Lactate Calcium Salt in Human Colorectal Cancer Cells.

Hypoxic cancer cells meet their growing energy requirements by upregulating glycolysis, resulting in increased glucose consumption and lactate production. Herein, we used a unique approach to change in anaerobic glycolysis of cancer cells by lactate calcium salt (CaLac). Human colorectal cancer (CRC) cells were used for the study. Intracellular calcium and lactate influx was confirmed following 2.5 mM CaLac treatment. The enzymatic activation of lactate dehydrogenase B (LDHB) and pyruvate dehydrogenase (PDH) through substrate reaction of CaLac was investigated. Changes in the intermediates of the tricarboxylic acid (TCA) cycle were confirmed. The cell viability assay, tube formation, and wound-healing assay were performed as well as the confirmation of the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). In vivo antitumor effects were evaluated using heterotopic and metastatic xenograft animal models with 20 mg/kg CaLac administration. Intracellular calcium and lactate levels were increased following CaLac treatment in CRC cells under hypoxia. Then, enzymatic activation of LDHB and PDH were increased. Upon PDH knockdown, α-ketoglutarate levels were similar between CaLac-treated and untreated cells, indicating that TCA cycle restoration was dependent on CaLac-mediated LDHB and PDH reactivation. CaLac-mediated remodeling of cancer-specific anaerobic glycolysis induced destabilization of HIF-1α and a decrease in VEGF expression, leading to the inhibition of the migration of CRC cells. The significant inhibition of CRC growth and liver metastasis by CaLac administration was confirmed. Our study highlights the potential utility of CaLac supplementation in CRC patients who display reduced therapeutic responses to conventional modes owing to the hypoxic tumor microenvironment.

Combination Antitumor Effect of Sorafenib via Calcium-Dependent Deactivation of Focal Adhesion Kinase Targeting Colorectal Cancer Cells.

Sorafenib has been recently used for the treatment of patients with advanced colorectal cancer (CRC) and is recognized for its therapeutic value. However, the continuous use of sorafenib may cause resistance in the treatment of cancer patients. In this study, we investigated whether sorafenib exerts an enhanced anticancer effect on CRC cells via the calcium-mediated deactivation of the focal adhesion kinase (FAK) signaling pathways. The appropriate dose of sorafenib and lactate calcium salt (CaLa) for a combination treatment were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Then, cell cycle analysis was performed following treatment with 2.5 µM sorafenib and/or 2.5 mM CaLa. CRC cells were found to be in the G1 phase by sorafenib treatment, and they accumulated in the sub-G1 phase with CaLa treatment. Western blots and enzyme-linked immunosorbent assays were performed to analyze the elements of the recombinant activated factor (RAF) and focal adhesion kinase (FAK) signaling cascades. Sorafenib-inhibited RAF-dependent signaling in CRC cells, however, either did not affect the expression of Akt or increased it. As the upstream signaling of FAK was suppressed by CaLa, we observed that the expression of the sub-signaling phospho (p) AKT and p-mammalian target of rapamycin was also suppressed. Treatment with a combination of sorafenib and CaLa enhanced the antitumor activity of CRC cells. The % viability of CRC cells was significantly decreased compared to the single treatment with sorafenib or CaLa, and the accumulation of Sub G1 of CRC cells was clearly confirmed. The migration ability of CRC cells was significantly reduced. The findings of this study indicate that sorafenib will show further improved antitumor efficacy against CRC due to overcoming resistance through the use of CaLa.

Synergistically Anti-metastatic Effect of 5-Flourouracil on Colorectal Cancer Cells via Calcium-mediated Focal Adhesion Kinase Proteolysis.

AIM: To investigate the possibility of enhancing an anti-metastatic effect of 5-fluorouracil (5-FU) on colorectal cancer (CRC) cells by combining it with continuous calcium supplementation. MATERIALS AND METHODS: Optimal doses of 5-FU with/without lactate salt (CaLa) were determined via clonogenicity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays using human CRC cells cultured on normal or low-attachment plates. Invasion and migration assays confirmed the enhanced anti-metastatic effect of combining 5-FU and CaLa. Western blot analysis for elements of the focal adhesion kinase (FAK) signaling cascade and epithelial-mesenchymal transition (EMT) markers was used to investigate the underlying mechanism. RESULTS: 5-FU (2.5 µM) had no antitumor activity against unanchored CRC cells, while it significantly suppressed anchorage-dependent cell proliferation. In contrast, treatment with CaLa (2.5 mM), alone and in combination with 5-FU, exerted antitumor activity against both anchored and unanchored CRC cells via calcium-mediated FAK proteolysis and inhibition of EMT markers, such as vimentin and SNAIL. CONCLUSION: Calcium supplementation represents a method of enhancing the potency of existing antitumor agents such as 5-FU, augmenting their clinical effectiveness.

Combination of lactate calcium salt with 5-indanesulfonamide and α-cyano-4-hydroxycinnamic acid to enhance the antitumor effect on HCT116 cells via intracellular acidification.

Maintenance of a neutral intracellular pH (pHi) is favorable for the survival of tumors, and maintenance of highly acidic extracellular pH (pHe) facilitates tumor invasiveness. The aim of the present study was to investigate the antitumor effects of lactate calcium salt (CaLa), 5-indanesulfonamide (IS) and α-cyano-4-hydroxycinnamic acid (CA) via pH regulation in colon cancer cells. HCT116 cells were treated with CaLa, IS, CA and combinations of the three. Subsequently, the concentration of intracellular lactate was determined. pHi and pHe were measured using cell lysates and culture media. Colony formation assay, cell viability assay and western blot analysis were additionally performed to analyze the consequences of the pH changes. CaLa, IS, CA and combination treatments induced an increase in the concentration of intracellular lactate. Lactate influx into the tumor microenvironment produced an acidic pHi in colon cancer cells. Consequently, colony formation and cell viability were significantly decreased, as well as poly(adenosine diphosphate-ribose) polymerase degradation. The tumor microenvironment may be exploited therapeutically by disrupting the mechanism that regulates pHi, leading to cell apoptosis. The present study indicated that treatment with CaLa, IS and CA induced intracellular acidification via lactate influx, causing apoptosis of colon cancer cells. Additionally, the findings suggested that the combination of CaLa with IS and CA may enhance antitumor activity, and may provide a potential therapeutic approach for the treatment of colon cancer.

Lactate calcium salt affects the viability of colorectal cancer cells via betaine homeostasis.

AIMS: Betaine plays an important role in cellular homeostasis. However, the physiological roles of betaine-γ-aminobutyric acid (GABA) transporter (BGT-1) are still being disputed in cancer. In this study, we tried to find the possibility of the antitumor effect on colorectal cancer (CRC) cell via lactate calcium salt (CaLa)-induced BGT-1 downregulation. MAIN METHODS: The CRC cell viability and clonogenic assay was performed using different doses of BGT-1 inhibitor. The expression level of BGT-1 was measured following the treatment of 2.5mM CaLa. Betaine was treated to confirm the resistance of the antitumor activity by CaLa. Tumor growth was also measured using a xenograft animal model. KEY FINDINGS: Long-term exposure of 2.5mM CaLa clearly decreased the expression of BGT-1 in the CRC cells. As a result of the downregulation of BGT-1 expression, the clonogenic ability of CRC cells was also decreased in the 2.5mM CaLa-treated group. Reversely, the number of colonies and cell viability was increased by combination treatment with betaine and 2.5mM CaLa, as compared with a single treatment of 2.5mM CaLa. Tumor growth was significantly inhibited in the xenograft model depending on BGT-1 downregulation by 2.5mM CaLa treatment. SIGNIFICANCE: These results support the idea that long-lasting calcium supplementation via CaLa contributes to disruption of betaine homeostasis in the CRC cells and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC.

Investigation of lactate calcium salt-induced ß-catenin destabilization in colorectal cancer cells.

AIMS: Calcium supplements appear to reduce the risk of developing colorectal cancer (CRC), and it is necessary to clarify the mechanisms by which they exert their effects. In the present study, we investigate the supplementation effect of calcium via lactate calcium salt (CaLa) on CRC cells, focusing on ß-catenin destabilization. MAIN METHODS: The clonogenic assay was performed using different doses of CaLa. The expression level of c-Myc and Cyclin D1 was measured in addition to the confirmation of ß-catenin expression in the CRC cells. Glycogen synthase kinase (GSK)-3ß expression was also confirmed in order to investigate the mechanism of ß-catenin degradation. Tumorigenic ability was confirmed using a xenograft animal model. KEY FINDINGS: The number of colonies was significantly decreased after 2.5mM CaLa treatment. CaLa-treated CRC cells showed a decrease in the ß-catenin expression. The quantitative level of the ß-catenin protein was significantly decreased in the CRC cell lysates, hence the expression level of c-Myc and cyclin D1 was significantly decreased following 2.5mM CaLa treatment. We also confirmed that an increased expression of GSK-3ß by CaLa is a key pathway in ß-catenin degradation. In the xenograft study, tumorigenicity was significantly inhibited to a maximum of 45% in the CaLa-treated group as compared with the control. SIGNIFICANCE: These results support the idea that calcium supplementation via CaLa contributes to ß-catenin degradation and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC.