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.

Abstract. 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 Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancerrelated death in both men and women (1). According to the estimates of the Global Cancer Control (GLOBOCAN) project, nearly 1.4 million people worldwide were diagnosed with CRC in 2012, with the Republic of Korea displaying the highest incidence rates, followed by Slovakia and Hungary (2).CRC that has grown into the wall can penetrate blood or lymph vessels; typically, cancer cells first invade nearby lymph nodes, and then spread to other parts of the body, such as the liver or lung (1, 3). Approximately 150,000 new cases are diagnosed each year, with about 20% of them already displaying metastases (2). Metastatic CRC is often harder to treat and tends to have a poor treatment outcome. As a result, patients with metastatic CRC have a low 5-year survival rate of about 11% (1, 2).In patients newly diagnosed with CRC, 5-fluorouracil (5-FU) is usually administered intravenously in combination with a second drug called leucovorin (4). Moreover, most patients with newly-diagnosed metastatic CRC undergo surgery after receiving chemotherapy with 5-FU (5). Other combinatorial treatments have also been tested for first-line chemotherapy of metastatic CRC, where a targeted drug, such as bevacizumab or cetuximab, is co-administered to increase the efficiency of 5-FU (6). However, these regimens suffer from increased toxicity, and cause symptoms such as neutropenia, severe diarrhea, and vomiting (7). Moreover, 5-FU is not an effective treatment strategy because it only delays micrometastasis. The mechanism through which 5-FU acts on CRC metastases remains unclear (8).Focal adhesion kinase (FAK), a protein kinase involved in cellular adhesion, is activated in several types of advancedstage cancer. In CRC cells, phosphorylation of FAK occurs on multiple residues, and overexpression of FAK has been detected in liver metastases of CRC (9). FAK has been implicated in the regulation of the expression of genes 103 Τhis article is freely accessible online.

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.

In this protocol, we introduced a method of measuring mitochondrial dysfunction to confirm the epithelial–mesenchymal transition (EMT) in pancreatic cancer cells under a hypoxic environment. There are many expertized and complicated methods to verify EMT. However, our methods have indicated that EMT can be identified by examining changes in reactive oxygen species (ROS) generation and membrane potential in mitochondria. To demonstrate whether the changes in the indicators of mitochondrial dysfunction are correlative to EMT, cell morphology, and expression of E-cadherin and N-cadherin were additionally observed. The results verified that a decrease in membrane potential and an increase in ROS in mitochondria were associated with EMT of pancreatic cancer cells. This protocol would be useful as a basis for providing an additional indicator for changes in the tumor microenvironment of pancreatic cancer cells relating to EMT under a hypoxic environment.

Abstract. 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.