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.

Challenges to addressing the unmet medical needs for immunotherapy targeting cold colorectal cancer.

With the establishment of the immune surveillance mechanism since the 1950s, attempts have been made to activate the immune system for cancer treatment through the discovery of various cytokines or the development of antibodies up to now. The fruits of these efforts have contributed to the recognition of the 3rd generation of anticancer immunotherapy as the mainstream of cancer treatment. However, the limitations of cancer immunotherapy are also being recognized through the conceptual establishment of cold tumors recently, and colorectal cancer (CRC) has become a major issue from this therapeutic point of view. Here, it is emphasized that non-clinical strategies to overcome the immunosuppressive environment and clinical trials based on these basic investigations are being made on the journey to achieve better treatment outcomes for the treatment of cold CRC.

Accumulation of poly (adenosine diphosphate-ribose) by sustained supply of calcium inducing mitochondrial stress in pancreatic cancer cells.

BACKGROUND: The biochemical phenomenon defined as poly adenosine diphosphate (ADP)-ribosylation (PARylation) is essential for the progression of pancreatic cancer. However, the excessive accumulation of poly ADP-ribose (PAR) induces apoptosis-inducing factor (AIF) release from mitochondria and energy deprivation resulting in the caspase-independent death of cancer cells. AIM: To investigate whether sustained calcium supply could induce an anticancer effect on pancreatic cancer by PAR accumulation. METHODS: Two pancreatic cancer cell lines, AsPC-1 and CFPAC-1 were used for the study. Calcium influx and mitochondrial reactive oxygen species (ROS) were observed by fluorescence staining. Changes in enzyme levels, as well as PAR accumulation and energy metabolism, were measured using assay kits. AIF-dependent cell death was investigated followed by confirming in vivo anticancer effects by sustained calcium administration. RESULTS: Mitochondrial ROS levels were elevated with increasing calcium influx into pancreatic cancer cells. Then, excess PAR accumulation, decreased PAR glycohydrolase and ADP-ribosyl hydrolase 3 levels, and energy deprivation were observed. In vitro and in vivo antitumor effects were confirmed to accompany elevated AIF levels. CONCLUSION: This study visualized the potential anticancer effects of excessive PAR accumulation by sustained calcium supply on pancreatic cancer, however elucidating a clear mode of action remains a challenge, and it should be accompanied by further studies to assess its potential for clinical application.

Inhibition of poly (ADP-Ribose) polymerase: A promising strategy targeting pancreatic cancer with BRCAness phenotype.

The use of chemotherapeutic regimens for the treatment of pancreatic cancer is still limited because pancreatic cancer is usually diagnosed at an advanced stage as a refractory disease in which symptoms are difficult to recognize in the early stages. Furthermore, at advanced stages, there are important challenges to achieve clinical benefit and symptom resolution, even with the use of an expanded spectrum of anticancer drugs. Recently, a point of reduced susceptibility to conventional chemotherapies by breast cancer susceptibility gene (BRCA) mutations led to a new perspective for overcoming the resistance of pancreatic cancer within the framework of increased genome instability. Poly (ADP-Ribose) polymerase (PARP) -1 is an enzyme that can regulate intrinsic functions, such as response to DNA damage. Therefore, in an environment where germline mutations in BRCAs (BRCAness) inhibit homologous recombination in DNA damage, resulting in a lack of DNA damage response, a key role of PARP-1 for the adaptation of the genome instability could be further emphasized. Here, we summarized the key functional role of PARP-1 in genomic instability of pancreatic cancer with the BRCAness phenotype and listed clinical applications and outcomes of PARP-1 inhibitors to highlight the importance of targeting PARP-1 activity.

Poly adenosine diphosphate-ribosylation, a promising target for colorectal cancer treatment.

The development of colorectal cancer (CRC) can result from changes in a variety of cellular systems within the tumor microenvironment. Particularly, it is primarily associated with genomic instability that is the gradual accumulation of genetic and epigenetic changes consisting of a characteristic set of mutations crucial for pathways in CRC progression. Based on this background, the potential to focus on poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP)-1 and poly-ADP ribosylation (PARylation) as the main causes of malignant formation of CRC may be considered. One of the important functions of PARP-1 and PARylation is its deoxyribonucleic acid (DNA) repair function, which plays a pivotal role in the DNA damage response and prevention of DNA damage maintaining the redox homeostasis involved in the regulation of oxidation and superoxide. PARP-1 and PARylation can also alter epigenetic markers and chromatin structure involved in transcriptional regulation for the oncogenes or tumor suppressor genes by remodeling histone and chromatin enzymes. Given the high importance of these processes in CRC, it can be considered that PARP-1 and PARylation are at the forefront of the pathological changes required for CRC progression. Therefore, this review addresses the current molecular biological features for understanding the multifactorial function of PARP-1 and PARylation in CRC related to the aforementioned roles; furthermore, it presents a summary of recent approaches with PARP-1 inhibition in non-clinical and clinical studies targeting CRC. This understanding could help embrace the importance of targeting PARP-1 and PARylation in the treatment of CRC, which may present the potential to identify various research topics that can be challenged both non-clinically and clinically.

The Significance of Targeting Poly (ADP-Ribose) Polymerase-1 in Pancreatic Cancer for Providing a New Therapeutic Paradigm.

Genome-wide studies focusing on elucidating the effects on cancer progression have enabled the consequent identification of a distinct subpopulation of pancreatic cancer cells with unstable genomic characteristics. Based on this background, deleterious changes by poly (adenosine diphosphate (ADP)-ribose) polymerase-1 (PARP)-1 have been concentrated in oncology. One of the critical functions of PARP-1 is the response to DNA damage, which plays a pivotal role in DNA repair in cancers. PARP-1 also has widespread functions that are essential for the survival and growth of cancer cells. It regulates oxidative stress in mitochondria through the regulation of superoxide and oxidation. PARP-1 is in charge of regulating mitosis, which is a crucial role in tumorigenesis and remodels histones and chromatin enzymes related to transcriptional regulation, causing alterations in epigenetic markers and chromatin structure. Given the significance of these processes, it can be understood that these processes in cancer cells are at the frontline of the pathogenetic changes required for cancer cell survival, and these contributions can result in malignant transformation. Therefore, this review addresses the current molecular biological features for understanding the multifactorial function of PARP-1 in pancreatic cancer related to the aforementioned roles, along with the summary of recent approaches with PARP-1 inhibition in clinical studies targeting pancreatic cancer. This understanding could help to embrace the importance of targeting PARP-1 in the treatment of pancreatic cancer, which may present the potential to find out a variety of research topics that can be both challenged clinically and non-clinically.

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.

Monitoring Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Investigation of Mitochondrial Dysfunction.

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.

Potential Anticancer Effect of Calcium-mediated Src Degradation on Hormone-dependent Breast Cancer.

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.

Cancer-specific metabolism: Promising approaches for colorectal cancer treatment.

Investigation of cancer-specific metabolism has made it possible to establish the principle that atypically reconstituted metabolism is considered a hallmark of cancer due to changes in physiological property. Recently, a variety of targets depending on the prompted aerobic glycolysis process, starting from the abnormal uptake of glucose, and cancer-specific metabolism due to impaired mitochondrial function and abnormal expression of drug-metabolizing enzymes have been investigated and discovered. Given that most solid cancers rely on cancer-specific metabolism to support their growth, it is necessary to examine closely the specific processes of cancer metabolism and have a detailed understanding of how cellular metabolism is altered in colorectal cancer (CRC) related to CRC survival and proliferation. The development of key methods to regulate efficiently cancer-specific metabolism in CRC is still in the initial stage. Therefore, targeting cancer-specific metabolism will yield treatable methods that are critical as a new area of development strategies for CRC treatment.

Investigation of the preventive effect of calcium on inflammation-mediated choroidal neovascularization.

AIMS: Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in elderly people. The pathogenesis of neovascular AMD is known but is closely related to inflammation and choroidal neovascularization (CNV). The aim of this study was to investigate the anti-inflammatory and anti-angiogenic effects of calcium on neovascular AMD. MAIN METHODS: Human retinal pigment epithelial cells (ARPE-19) were used to identify protein markers of inflammation induced by differentiated macrophages. Choroidal neovascularization (CNV) mouse model was established by rupturing the Bruch's membrane using laser photocoagulation in C57BL/6 mice. Mice were divided into the following groups: untreated control and calcium supplemented. The expression levels of toll-like receptor isotype (TLR) 4, nuclear factor kappa B (NF-κB), hypoxia-inducible factor-1α (Hif-1α), and vascular endothelial growth factor (VEGF) were investigated to check whether calcium supplementation results in suppression of inflammation and has an anti-angiogenic effect. CNV was evaluated by immunofluorescence staining on choroidal flat mounts. KEY FINDING: The inflammation-induced expression of TLR4, NF-κB, and Hif-1α was decreased in ARPE-19 cells after calcium supplementation. Inhibition of the transcriptional activation of ARPE-19 cells by Hif-1α suppression resulted in decreased VEGF expression. In the laser-induced CNV mouse model, calcium supplementation inhibited inflammatory mediators and neovascularization in the retinal tissue. SIGNIFICANCE: Supplementation with calcium seems to constrain inveterate symptoms of neovascular AMD by inhibiting inflammation and angiogenesis in the laser-induced CNV mouse model.

Inhibiting focal adhesion kinase: A potential target for enhancing therapeutic efficacy in colorectal cancer therapy.

Focal adhesion kinase (FAK) is a major integrin-dependent tyrosine phosphorylated protein, recently, FAK association with colorectal cancer (CRC) has gained attention. The various cancer-promoting mechanisms that associated with FAK can be implicated in the progression of CRC. The interactions between structural features of FAK and various kinases could be closely related to growth, survival, and metastasis in CRC cells. These interactions include human epithelial growth factor receptor, c-Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and Src. Such interactions can trigger the survival signaling of CRC cells and are also involved signaling downstream of phosphatidylinositol 3-kinase, AKT, and the extracellular regulated kinase. Based on this scientific background, many pharmaceutical companies are taking efforts to develop FAK inhibitors to treat solid cancer including CRC. Although the anti-cancer efficacies have been noted in many studies, the commercial drugs have not been developed yet. Therefore, the FAK research on CRC is expected to gain momentum and be highly appreciated as a potential field for developing the new drugs. Therefore, the studies on FAK that effect on the progression of human CRC s would be possible to suggest various approaches to CRC treatment, and FAK could be a potential target as an anticancer candidate for CRC therapies.

Investigation into Enhancing Capecitabine Efficacy in Colorectal Cancer by Inhibiting Focal Adhesion Kinase Signaling.

BACKGROUND/AIM: Capecitabine is a pro-drug of 5-fluorouracil (5-FU), and is an orally available chemotherapeutic used to treat colorectal cancer (CRC). Recently, research has focused on improving its efficacy at lower doses in order to minimize its well-known toxicities. In this study, we investigated the possibility of improving the antitumor effect of capecitabine against CRC by destabilizing focal adhesion kinase (FAK) signaling. MATERIALS AND METHODS: Optimal dosages for capecitabine and lactate calcium salt (LCS) were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide MTT assay. The viability of CRC cells was investigated by MTT and clonogenic assays after single or combination treatment with capecitabine and LCS. Western blot analyses were used to determine changes in the expression of components of the FAK and AKT signaling cascade, and this information was used to elucidate the underlying mechanism. A xenograft model was established to evaluate the antitumor efficacy of the combination treatment, as well as its necrotic effect and organ toxicity. RESULTS: The addition of LCS to capecitabine treatment led to an increase in the proteolysis of the FAK signaling cascade components, including SRC proto-oncogene, non-receptor tyrosine kinase; AKT serine/threonine kinase 1; and nuclear factor-kappa B, resulting in a decrease in the viability and clonogenic ability of CRC cells. In vivo antitumor efficacy, including tumor necrosis, was significantly increased with the combination treatment relative to both single treatments, and no organ toxicity was found in any experimental group. CONCLUSION: The addition of LCS increased the anticancer efficacy of capecitabine at a lower dose than is currently used in human patients.

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.

Perspective on Cancer Therapeutics Utilizing Analysis of Circulating Tumor Cells.

Various methods are available for cancer screening, and the methods are performed depending on the origin site of cancer. Among these methods, biopsy followed by medical imaging is the most common. After cancer progression is determined, an optimal treatment-such as surgery, chemotherapy, and/or radiation therapy-is selected. A new assay has been developed that detects circulating tumor cells (CTCs). Tracking changes in CTCs may reveal important tumoral sensitivity information or resistance patterns to specific regimens and prompt changes in therapy on a personalized basis. Characterization of CTCs at the DNA, RNA, and protein levels is important for gaining insight for clinical applications. A small number of CTCs can be analyzed to obtain genome information such as the progression of cancer including metastasis, even in a single cluster. Although many clinical studies, particularly CTC enumeration and detection of specific oncogene expression, have increased the success rate of diagnosis and predicting prognosis, there is no consensus regarding the technical approaches and various aspects of the methodology, making it difficult to standardize optimal methods for CTC analysis. However, ongoing technological advances are currently being achieved and large-scale clinical studies are being conducted. Applying CTC analysis in the clinic would be very useful for advancing diagnosis, prognosis prediction, and therapeutics.

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.

Enhancement of the Antitumor Effect of Methotrexate on Colorectal Cancer Cells via Lactate Calcium Salt Targeting Methionine Metabolism.

Methionine (Met) is involved in one-carbon de novo nucleotide synthesis and is an essential amino acid for cell survival. The impact of lactate calcium salt (CaLa) on the Met metabolism was investigated to evaluate the enhanced antitumor effect of methotrexate (MTX) on colorectal cancer (CRC) cells. Met dependency relating to homocysteine (Hcy) and betaine was investigated in human CRC cells (HCT-116 and HT-29) using a viability assay and liquid chromatography-mass spectrometry. Expression of betaine transporter-1 (BGT-1) following treatment with MTX alone or with CaLa was determined by Western blot. Enhanced antitumor effect due to malfunction of Met synthesis was confirmed. CRC cell viability decreased in Met-restricted medium, but was maintained after Hcy and betaine treatment while overcoming Met restriction. BGT-1 expression was downregulated following the treatment of dose-increased CaLa, whereas there was no effect on BGT-1 expression after MTX treatment. CaLa in combination with MTX induced reduced Met synthesis when CRC cell viability was reduced. The results indicated that CaLa-mediated BGT-1 downregulation inhibits Met synthesis by disrupting betaine homeostasis. CaLa raised the antitumor effect of MTX via secondary role in the inhibition of the de novo nucleotide synthesis. Combination therapy of MTX and CaLa could maximize the effectiveness of CRC treatment.

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.