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.

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.

Changes in TRPV1-Mediated Physiological Function in Rats Systemically Treated With Capsaicin on the Neonate.

Capsaicin is the active component of chili peppers and is a hydrophobic, colorless, odorless, and crystalline to waxy compound. The transient receptor potential vanilloid 1 (TRPV1) is the capsaicin receptor channels that are involved in a variety of functions like transduction and transmission of the physiological stimulus. Subcutaneous injection of capsaicin to a newborn rat leads to involuntary lifelong TRPV1 desensitization. Various physiological changes including sensory and homeostatic actions in the body associated with neonatal capsaicin treatment are induced by direct TRPV1 channel targeting. Interesting changes include unique phenomena such as the reduction in pain perception, abnormal body temperature, increase in infection, infectious or neuropathological itching, and irregular circadian core body temperature rhythm. These symptoms are associated with relatively higher fever or loss of sensory c-fiber related to TRPV1 desensitization. The aforementioned outcomes not only provide a warning about the risk of capsaicin exposure in newborns but also indicate the possible occurrence of relatively rare diseases that are difficult to diagnose. Therefore, Therefore, the present review aims to summarize the unique phenomena caused by systemic capsaicin administration in neonatal rats.

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.

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.

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.

Investigation of the functional difference between the pathological itching and neuropathic pain-induced rat brain using manganese-enhanced MRI.

BACKGROUND: There is a remarkable similarity in the central sensitization of itch and pain. However, the interactions between itch and pain are only partially understood. PURPOSE: To investigate the functional activity of cerebral regions to provide clear information on the neuronal pathways related to both pathological itching (PI) and neuropathic pain (NP). MATERIAL AND METHODS: Sprague-Dawley rats were used in this study. PI was induced via neonatal capsaicin treatment, and scratching behavior was counted. NP was induced via lumbar spinal nerve 5 (L5) ligation, and mechanical allodynia was measured. The activated cerebral regions in the control, PI, and NP rats were measured using a 4.7 T magnetic resonance imaging (MRI) system and manganese-enhanced MRI (MEMRI). Subsequently, the cerebral activation regions were identified, and the signal intensity was compared. RESULTS: Cerebral activities of the PI-induced rats were found in three regions -7.10 and -4.20 mm, and two regions -2.45 mm from the bregma. In the NP-induced rats, cerebral activities were found in two regions 7.10 and -2.45 mm, and one region -4.20 mm from the bregma. Comparing the PI and NP rats, the cerebral activities were different in one region -7.10 mm and -2.45 mm, and two regions -4.20 mm from the bregma. The different regions were the midbrain area, the geniculate complex, the hypothalamic area, and the amygdala area. CONCLUSION: Our MEMRI investigation indicates functionally different activity of cerebral regions due to the effect of PI or NP. These findings provide clear information of the signal transduction in the brain regarding PI or NP that share a similar neuronal pathway.

Irradiation-induced localization of IL-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma.

Irradiation in conjunction with gene therapy is considered for efficient cancer treatment. Mesenchymal stem cells (MSCs), due to their irradiation-promotable tumor tropism, are ideal delivery vehicles for gene therapy. In this study, we investigated whether treatment with radiation and interleukin (IL)-12-expressing MSCs (MSCs/IL-12) exerts improved antitumor effects on murine metastatic hepatoma. HCa-I and Hepa 1-6 cells were utilized to generate heterotopic murine hepatoma models. Tumor-bearing mice were treated with irradiation or MSCs/IL-12 alone, or a combination. Monocyte chemoattractant protein-1 (MCP-1/CCL2) expression was assessed in irradiated hepatoma tissues to confirm a chemotactic effect. Combination treatment strategies were established and their therapeutic efficacies were evaluated by monitoring tumor growth, metastasis and survival rate. IL-12 expression was assessed and the apoptotic activity and immunological alterations in the tumor microenvironment were examined. MCP-1/CCL2 expression and localization of MSCs/IL-12 increased in the irradiated murine hepatoma cells. The antitumor effects, including suppression of pulmonary metastasis and survival rate improvements, were increased by the combination treatment with irradiation and MSCs/IL-12. IL-12 expression was increased in tumor cells, causing proliferation of cluster of differentiation 8(+) T-lymphocytes and natural killer cells. The apoptotic activity increased, indicating that the cytotoxicity of immune cells was involved in the antitumor effect of the combined treatment. Treatment with irradiation and MSCs/IL-12 showed effectiveness in treating murine metastatic hepatoma. IL-12-induced proliferation of immune cells played an important role in apoptosis of tumor cells. Our results suggest that treatment with irradiation and MSCs/IL-12 may be a useful strategy for enhancing antitumor activity in metastatic hepatoma.

Investigation of the pruritus-induced functional activity in the rat brain using manganese-enhanced MRI.

PURPOSE: To provide clear information on the activation regions of itching, we investigated the functional activity of cerebral regions in the pruritus-induced rat model using manganese-enhanced magnetic resonance imaging (MEMRI). MATERIALS AND METHODS: Itching was induced via neonatal capsaicin treatment in Sprague-Dawley rats (itching rats), and scratching behavior of the control, itching, and gabapentin (GBP)-treated itching rats was compared. Then the activated or deactivated brain regions were investigated in the control, itching, and GBP-treated itching rats using a 4.7T MRI system. RESULTS: While the itching rats engaged in vigorous scratching (121.2 ± 22.4 times), the scratching behavior was decreased in the GBP-treated itching rats (30.6 ± 8.8 times). GBP induced the attenuation of functional activity in two regions -7.10 mm from bregma, in one region -6.65 mm from bregma, and in one region -6.06 mm from bregma. The brain regions related to itching were as follows: parafascicular nucleus, thalamus, superior/inferior colliculus, periaqueductal gray, cingulate cortex, amygdala, midbrain regions, lateral habenula, and hypothalamic areas. CONCLUSION: Our MEMRI investigation indicates new functional activity of cerebral regions in rats due to the effect of itching or GBP. This information could be used to monitor the therapeutic effects of novel agents or for clinical strategies to treat pathological itch.

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.

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.

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.

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.

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.