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.

Antitumor Effect of Calcium-Mediated Destabilization of Epithelial Growth Factor Receptor on Non-Small Cell Lung Carcinoma.

Despite the development of numerous therapeutics targeting the epithelial growth factor receptor (EGFR) for non-small cell lung carcinoma (NSCLC), the application of these drugs is limited because of drug resistance. Here, we investigated the antitumor effect of calcium-mediated degradation of EGFR pathway-associated proteins on NSCLC. First, lactate calcium salt (LCS) was utilized for calcium supplementation. Src, α-tubulin and EGFR levels were measured after LSC treatment, and the proteins were visualized by immunocytochemistry. Calpeptin was used to confirm the calcium-mediated effect of LCS on NSCLC. Nuclear expression of c-Myc and cyclin D1 was determined to understand the underlying mechanism of signal inhibition following EGFR and Src destabilization. The colony formation assay and a xenograft animal model were used to confirm the in vitro and in vivo antitumor effects, respectively. LCS supplementation reduced Src and α-tubulin expression in NSCLC cells. EGFR was destabilized because of proteolysis of Src and α-tubulin. c-Myc and cyclin D1 expression levels were also reduced following the decrease in the transcriptional co-activation of EGFR and Src. Clonogenic ability and tumor growth were significantly inhibited by LSC treatment-induced EGFR destabilization. These results suggest that other than specifically targeting EGFR, proteolysis of associated molecules such as Src or α-tubulin may effectively exert an antitumor effect on NSCLC via EGFR destabilization. Therefore, LCS is expected to be a good candidate for developing novel anti-NSCLC therapeutics overcoming chemoresistance.

Enhancing 5-Fluorouracil Efficacy in a Primary Colorectal Cancer by Long-lasting Calcium Supplementation.

BACKGROUND/AIM: 5-Fluorouracil (5-FU) over-use has led to an urgent need for alternative treatment regimens, such as a lower concentration of the drug because of its toxic effects. The aim of this study was to investigate the possibility of improving the antitumor effect of 5-FU without toxicity by targeting primary colorectal cancer (CRC) with sustained calcium supplementation. MATERIALS AND METHODS: The viability of CRC cells was determined after treatment of 5-FU, lactate calcium salt (CaLac), or the combination of te two. Western blot analysis for the focal adhesion kinase (FAK) signaling cascade was performed to investigate the underlying mechanism. A xenograft model was established to evaluate antitumor efficacy of each treatment, and the necrotic effect was also observed in tumor tissues. RESULTS: By the combined treatment, proteolysis of FAK signaling cascade, was mediated by sustained calcium supplementation resulting in further decrease in the clonogenicity of CRC cells. The in vivo anticancer efficacy including tumor necrosis was significantly increased by the combination treatment compared to single treatment of with 5-FU. CONCLUSION: Sustained calcium supplementation was able to enhance the potency of 5-FU targeting the primary CRC.

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.

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.

Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain.

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.