Selenium modulates AR/IGF-1R/EGFR and TROP2 signaling pathways and improves anticancer efficacy in murine mammary carcinoma 4T1.

The micronutrient selenium (Se) has been shown to exert potential anticancer properties. This study aimed to evaluate the effects of Se (in Se yeast form) on the selenoproteins (SELENO), AR/IGF-1R/EGFR, PI3K/Akt/mTOR and Ras/Raf/ERK cascades, and immune checkpoint blockade in TNBC murine 4T1 cells. We also assessed the effects of combination treatment with chemotherapeutic doxorubicin and Se on trophoblast cell surface antigen 2 (TROP2) levels. Compared with the control groups, cells incubated with Se (0.25, 0.5, 0.75, 1.0, 1.5 µg Se/mL) have lower viability, raised intracellular Se concentrations and SELENO expression, and higher malondialdehyde products in a dose-dependent manner. Se induced the inactivation of AR/IGF-1R/EGFR and downregulation of the PI3K/Akt/mTOR and Ras/Raf/ERK signaling molecules. Se-treated cells also exhibited decreased mitochondrial membrane potential, reduced levels of the cell cycle regulatory protein cyclin D1, cancer stemness, metastatic and EMT-related markers, and increased apoptosis. Subsequently, Se treatment significantly suppressed PD-1/PD-L1 and CTLA-4 mRNA levels and proteins. Doxorubicin decreased 4T1 cell viability and TROP2 expression levels, but the addition of Se to doxorubicin contributed to further reductions. Similar responses to Se treatment were also observed in the human MDA-MB-231 and MCF-7 breast cancer cells. These results show that Se upregulates SELENO and anti-AR/IGF-1R/EGFR signaling in TNBC cells, thus inducing oxidative stress-dependent apoptosis and cell cycle arrest, stemness, EMT, and metastasis, as well as blocking the immune checkpoint molecules. TROP2 down-regulation with Se is also a potential anti-TNBC therapeutic target.

Targeting EGFR in Combination with Nutritional Supplements on Antitumor Efficacy in a Lung Cancer Mouse Model.

Selenium (Se) and fish oil (FO) exert anti-epidermal growth factor receptor (EGFR) action on tumors. This study aimed to compare the anti-cancer efficacy of EGFR inhibitors (gefitinib and erlotinib) alone and in combination with nutritional supplements of Se/FO in treating lung cancer. Lewis LLC1 tumor-bearing mice were treated with a vehicle or Se/FO, gefitinib or gefitinib plus Se/FO, and erlotinib or erlotinib plus Se/FO. The tumors were assessed for mRNA and protein expressions of relevant signaling molecules. Untreated tumor-bearing mice had the lowest body weight and highest tumor weight and volume of all the mice. Mice receiving the combination treatment with Se/FO and gefitinib or erlotinib had a lower tumor volume and weight and fewer metastases than did those treated with gefitinib or erlotinib alone. The combination treatment exhibited greater alterations in receptor signaling molecules (lower EGFR/TGF-ß/TßR/AXL/Wnt3a/Wnt5a/FZD7/ß-catenin; higher GSK-3ß) and immune checkpoint molecules (lower PD-1/PD-L1/CD80/CTLA-4/IL-6; higher NKp46/CD16/CD28/IL-2). These mouse tumors also had lower angiogenesis, cancer stemness, epithelial to mesenchymal transitions, metastases, and proliferation of Ki-67, as well as higher cell cycle arrest and apoptosis. These preliminary results showed the Se/FO treatment enhanced the therapeutic efficacies of gefitinib and erlotinib via modulating multiple signaling pathways in an LLC1-bearing mouse model.

Fish Oil and Selenium with Doxorubicin Modulates Expression of Fatty Acid Receptors and Selenoproteins, and Targets Multiple Anti-Cancer Signaling in Triple-negative Breast Cancer Tumors.

Omega-3 fatty acids from fish oil (FO) and selenium (Se) potentiate some conventional therapies and have anticancer immune potential. This study aims to determine whether FO/Se modulates G-protein-coupled polyunsaturated fatty acid receptors (GPR-40 and GPR-120) and selenoproteins (Sel-H, Sel-W, and GPx4), and increases the therapeutic effect of doxorubicin in a dose-dependent manner on triple-negative breast cancer (TNBC) mouse. Mice were randomized into 5 groups (n = 7/group) and treated with physiological saline (control), low-dose doxorubicin, and doxorubicin in combination with low, medium, or high doses of FO/Se. The expression of signaling molecules in tumors was determined by measuring either mRNA or protein expression. Compared with doxorubicin alone, combination treatment resulted in lower tumor sizes and fewer overall metastasis, lower GPR-40 mRNA levels, and higher expression of all selenoproteins. Doxorubicin-FO/Se combination treatment decreased expression of membrane EGFR and FGFR, down-regulated downstream PI3K/AKT/mTOR, MAPK/ERK, and JAK2/c-Src/STAT3 signaling, increased tumor suppressor PTEN/TSC1/TSC2 expression and P53 activation, and suppressed oncogenic transcription factor expression. Dose-dependent inhibition of proliferation index Ki-67, cell cycle, and stem-cell-related markers were observed. Decreased immune check-points PD-L1/CTLA-4/Foxp3/CD86 and increased PD-1/CD28/IL-2 expression was also found. These observations suggest that the nutritional supplements FO/Se increase the chemotherapeutic efficacy of doxorubicin against TNBC by modulating GPR-40 and selenoprotein and targeting multiple signaling pathways in tumor tissues.

Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells.

Cisplatin is a prevalent chemotherapeutic agent used for non-small cell lung cancer (NSCLC) that is difficult to treat by targeted therapy, but the emergence of resistance severely limits its efficacy. Thus, an effective strategy to combat cisplatin resistance is required. This study demonstrated that, at clinically achievable concentrations, the combination of selenium yeast (Se-Y) and fish oil (FO) could synergistically induce the apoptosis of cancer stem cell (CSC)-like A549 NSCLC sphere cells, accompanied by a reversal of their resistance to cisplatin. Compared to parental A549 cells, sphere cells have higher cisplatin resistance and possess elevated CSC markers (CD133 and ABCG2), epithelial-mesenchymal transition markers (anexelekto (AXL), vimentin, and N-cadherin), and cytoprotective endoplasmic reticulum (ER) stress marker (glucose-regulated protein 78) and increased oncogenic drivers, such as yes-associated protein, transcriptional coactivator with PDZ-binding motif, ß-catenin, and cyclooxygenase-2. In contrast, the proapoptotic ER stress marker CCAAT/enhancer-binding protein homologous protein and AMP-activated protein kinase (AMPK) activity were reduced in sphere cells. The Se-Y and FO combination synergistically counteracted the above molecular features of A549 sphere cells and diminished their elevated CSC-like side population. AMPK inhibition by compound C restored the side population proportion diminished by this nutrient combination. The results suggest that the Se-Y and FO combination can potentially improve the outcome of cisplatin-treated NSCLC with phenotypes such as A549 cells.

Fish Oil, Se Yeast, and Micronutrient-Enriched Nutrition as Adjuvant Treatment during Target Therapy in a Murine Model of Lung Cancer.

Despite the effectiveness of primary treatment modalities for cancer, the side effects of treatments, medication resistance, and the deterioration of cachexia after disease progression lead to poor prognosis. A supportive treatment modality to overcome these limitations would be considered a major breakthrough. Here, we used two different target drugs to demonstrate whether a nutraceutical formula (fish oil, Se yeast, and micronutrient-enriched nutrition; NuF) can interfere with cancer cachexia and improve drug efficacy. After Lewis lung cancer (LLC) tumor injection, the C57BL/6 mice were orally administered targeted therapy drugs Iressa and Sutent alone or combined with NuF for 27 days. Sutent administration effectively inhibited tumor size but increased the number of lung metastases in the long term. Sutent combined with NuF had no significant difference in tumor weight and metastasis compare with Sutent alone. However, NuF slightly attenuated metastases number in lung may via mesenchymal marker N-cadherin suppression. NuF otherwise increased epithelial-like marker E-cadherin expression and induce NO-mediated intrinsic apoptotic pathway in tumor cells, thereby strengthening the ability of the targeted therapy drug Iressa for inhibiting tumor progression. Our results demonstrate that NuF can promote the anticancer effect of lung cancer to targeted therapy, especially in Iressa, by inhibiting HIF-1α and epithelial-mesenchymal transition (EMT) and inducing the apoptosis of lung cancer cells. Furthermore, NuF attenuates cancer-related cachectic symptoms by inhibiting systemic oxidative stress.

Nutritional supplements in combination with chemotherapy or targeted therapy reduces tumor progression in mice bearing triple-negative breast cancer.

The potential anti-cancer properties of selenium (Se) and eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) have been documented. However, few studies have been conducted examining anti-tumor effects of nutritional supplements (NS) containing Se and EPA/DHA in combination with anti-cancer agents, such as taxol (Tax), adriamycin (Adr), and avastin (Ava). Compared with triple-negative breast cancer (TNBC)-bearing positive control (TB) mice, a low dose of Tax, Adr, and Ava decreased tumor size and the incidence of metastasis in TB-Tax, TB-Adr, and TB-Ava groups. Combination treatment with anti-cancer agent and NS (2.7 µg Se and 5.1 mg EPA/3.7 mg DHA/g) induced additional decreases in TB-Tax-NS, TB-Adr-NS, and TB-Ava-NS groups. Th1-associated cytokines were increased, and Th2-type cytokines were decreased significantly in TB mice with combination treatment than that of anti-cancer agent treatment alone. Combination treatment with anti-cancer agents and NS has also been shown to further increased tumor malondialdehyde (MDA) levels, lowered hypoxia-inducible factor (HIF)-1α, angiogenic markers (vascular endothelial growth factor [VEGF] and CD31) and metastatic potential, as well as reduced heat shock proteins, receptor tyrosine kinase AXL, and surface markers of cancer stem cells, and increased apoptotic proteins. For immune checkpoint molecules, combination treatment was associated with a greater decrease in programmed cell death ligand-1 (PD-L1) in both tumors and mammary glands, but PD-1 level in primary tumors was increased. Our results suggest that combination treatment with low-dose anti-cancer agents (Tax, Adr, and Ava) and oral supplementation of Se/ EPA/DHA significantly decreased tumor growth and metastatic progression in TNBC mice through multiple anti-tumor mechanisms.

Omega-3 Fatty Acid-Enriched Fish Oil and Selenium Combination Modulates Endoplasmic Reticulum Stress Response Elements and Reverses Acquired Gefitinib Resistance in HCC827 Lung Adenocarcinoma Cells.

Non-small cell lung cancer (NSCLC)-carrying specific epidermal growth factor receptor (EGFR) mutations can be effectively treated by a tyrosine kinase inhibitor such as gefitinib. However, the inevitable development of acquired resistance leads to the eventual failure of therapy. In this study, we show the combination effect of omega-3 fatty acid-enriched fish oil (FO) and selenium (Se) on reversing the acquired gefitinib-resistance of HCC827 NSCLC cells. The gefitinib-resistant subline HCC827GR possesses lowered proapoptotic CHOP (CCAAT/enhancer-binding protein homologous protein) and elevated cytoprotective GRP78 (glucose regulated protein of a 78 kDa molecular weight) endoplasmic reticulum (ER) stress response elements, and it has elevated ß-catenin and cyclooxygenase-2 (COX-2) levels. Combining FO and Se counteracts the above features of HCC827GR cells, accompanied by the suppression of their raised epithelial-to-mesenchymal transition (EMT) and cancer stem markers, such as vimentin, AXL, N-cadherin, CD133, CD44, and ABCG2. Accordingly, an FO and Se combination augments the gefitinib-mediated growth inhibition and apoptosis of HCC827GR cells, along with the enhanced activation of caspase -3, -9, and ER stress-related caspase-4. Intriguingly, gefitinib further increases the elevated ABCG2 and cancer stem-like side population in HCC827GR cells, which can also be diminished by the FO and Se combination. The results suggest the potential of combining FO and Se in relieving the acquired resistance of NSCLC patients to targeted therapy.

Antitumor, Inhibition of Metastasis and Radiosensitizing Effects of Total Nutrition Formula on Lewis Tumor-Bearing Mice.

Non-small-cell lung cancer (NSCLC) causes high mortality. Radiotherapy is an induction regimen generally applied to patients with NSCLC. In view of therapeutic efficacy, the outcome is not appealing in addition to bringing about unwanted side effects. Total nutrition is a new trend in cancer therapy, which benefits cancer patients under radiotherapy. Male C57BL/6JNarl mice were experimentally divided into five groups: one control group, one T group (borne with Lewis lung carcinoma but no treatment), and three Lewis lung carcinoma-bearing groups administrated with a total nutrition formula (T + TNuF group), a local radiotherapy plus daily 3 Gy in three fractions (T + R group), or a combination TNuF and radiotherapy (T + R + TNuF group). These mice were assessed for their mean tumor volumes, cachectic symptoms and tumor metastasis. TNuF administration significantly suppressed tumor growth and activated apoptotic cell death in NSCLC-bearing mice under radiation. The body-weight gain was increased, while the radiation-induced cachexia was alleviated. Analysis of mechanisms suggests that TNuF downregulates EGFR and VEGF signaling pathways, inhibiting angiogenesis and metastasis. In light of radiation-induced tumor cell death, mitigation of radiation-induced cachexia and inhibition of tumor cell distant metastasis, the combination of TNuF and radiotherapy synergistically downregulates EGFR and VEGF signaling in NSCLC-bearing mice.

Opposite Regulation of CHOP and GRP78 and Synergistic Apoptosis Induction by Selenium Yeast and Fish Oil via AMPK Activation in Lung Adenocarcinoma Cells.

Selenium has been intensively studied for the use of cancer prevention and treatment. However, the clinical effects are still plausible. To enhance its efficacy, a combinational study of selenium yeast (SY) and fish oil (FO) was performed in A549, CL1-0, H1299, HCC827 lung adenocarcinoma (LADC) cells to investigate the enhancement in apoptosis induction and underlying mechanism. By sulforhodamine B staining, Western blot and flow cytometric assays, we found a synergism between SY and FO in growth inhibition and apoptosis induction of LADC cells. In contrast, the fetal lung fibroblast cells (MRC-5) were unsusceptible to this combination effect. FO synergized SY-induced apoptosis of A549 cells, accompanied with synergistic activation of AMP-activated protein kinase (AMPK) and reduction of Cyclooxygenase (COX)-2 and ß-catenin. Particularly, combining with FO not only enhanced the SY-elevated proapoptotic endoplasmic reticulum (ER) stress marker CCAAT/enhancer-binding protein homologous protein (CHOP), but also reduced the cytoprotective glucose regulated protein of molecular weight 78 kDa (GRP78). Consequently, the CHOP downstream targets such as phospho-JNK and death receptor 5 were also elevated, along with the cleavage of caspase-8, -3, and the ER stress-related caspase-4. Accordingly, inhibition of AMPK by compound C diminished the synergistic apoptosis induction, and elevated CHOP/GRP78 ratio by SY combined with FO. The AMPK-dependent synergism suggests the combination of SY and FO for chemoprevention and integrative treatment of LADC.

Supplementation with Selenium yeast on the prooxidant-antioxidant activities and anti-tumor effects in breast tumor xenograft-bearing mice.

Selenium (Se) is essential for antioxidant activity involved in immune function and anti-carcinogenic action, whereas at higher concentrations, Se may have pro-oxidant properties. The present study was aimed at determining the effects of Se supplementation, as Se yeast, on oxidative stress in non-tumor/tumor tissues, as well as regulation of the apoptotic process, and immune responses in mice-bearing breast tumor xenografts. Female BALB/cByJNarl mice were divided into control (CNL and CNL-con), Se-supplemented control (CNL-HS, given as a single oral dose of 912 ng Se daily), breast tumor-bearing (TB and TB-con), TB-LS (228 ng Se), TB-MS (456 ng Se) and TB-HS (912 ng Se) groups. All mice were treated with/without Se for 14 days. A number of variables were further measured. Compared with the TB groups, tumor bearing mice with Se supplement had increased plasma Se concentrations, reduced erythrocyte Se-dependent glutathione peroxidase (GPx) activity and malondialdehyde (MDA) products and inhibited tumor growth. They have also higher Se concentrations in non-tumor and tumor tissues. Significantly elevated concentrations of MDA and reduced GPx activities, as well as increased anti-apoptotic bcl-2 and tumor suppressor p53 concentrations in tumor tissues were observed as Se accumulated in tumor, whereas lower MDA products were found in various non-tumor tissues than did the corresponding values. Further, there were elevated concentrations of Th1-derived cytokines and decreased Th2-type interleukin (IL)-4 in tumor-bearing mice with the treatment of Se. In conclusion, accumulation of Se in tumors may induce oxidative stress and p53-dependent pro-oxidative apoptosis, thus inhibiting the growth of breast tumor.

Effects of Selenium Yeast on Oxidative Stress, Growth Inhibition, and Apoptosis in Human Breast Cancer Cells.

Recent evidence suggests that selenium (Se) yeast may exhibit potential anti-cancer properties; whereas the precise mechanisms remain unknown. The present study was aimed at evaluating the effects of Se yeast on oxidative stress, growth inhibition, and apoptosis in human breast cancer cells. Treatments of ER-positive MCF-7 and triple-negative MDA-MB-231 cells with Se yeast (100, 750, and 1500 ng Se/mL), methylseleninic acid (MSA, 1500 ng Se/mL), or methylselenocysteine (MSC, 1500 ng Se/mL) at a time course experiment (at 24, 48, 72, and 96 h) were analyzed. Se yeast inhibited the growth of these cancer cells in a dose- and time-dependent manner. Compared with the same level of MSA, cancer cells exposure to Se yeast exhibited a lower growth-inhibitory response. The latter has also lower superoxide production and reduced antioxidant enzyme activities. Furthermore, MSA (1500 ng Se/mL)-exposed non-tumorigenic human mammary epithelial cells (HMEC) have a significant growth inhibitory effect, but not Se yeast and MSC. Compared with MSA, Se yeast resulted in a greater increase in the early apoptosis in MCF-7 cells as well as a lower proportion of early and late apoptosis in MDA-MB-231 cells. In addition, nuclear morphological changes and loss of mitochondrial membrane potential were observed. In conclusion, a dose of 100 to 1500 ng Se/mL of Se yeast can increase oxidative stress, and stimulate growth inhibitory effects and apoptosis induction in breast cancer cell lines, but does not affect non-tumorigenic cells.

Skeletal muscle atrophy is attenuated in tumor-bearing mice under chemotherapy by treatment with fish oil and selenium.

Chemotherapy can cause cachexia, which is manifested by weight loss, inflammation and muscle atrophy. However, the mechanisms of tumor and chemotherapy on skeletal muscle proteolysis, remained unclear. In this report, we demonstrated that tumor-induced myostatin in turn induced TNF-α, thus activating calcium-dependent and proteasomal protein degradation. Chemotherapy activated myostatin-mediated proteolysis and muscle atrophy by elevating IL-6. In tumor-bearing mice under chemotherapy, supplementation with fish oil and selenium prevented a rise in IL-6, TNF-α and myostatin and muscle atrophy. The findings presented here allow us to better understand the molecular basis of cancer cachexia and potentiate nutrition supplementation in future cancer chemotherapy.

Omega-3 fatty acid-, micronutrient-, and probiotic-enriched nutrition helps body weight stabilization in head and neck cancer cachexia.

OBJECTIVE: To evaluate whether an oral nutritional supplement enriched with omega-3 fatty acids, micronutrients, and probiotics affected body weight (BW) changes, serum albumin and prealbumin levels in patients with head and neck cancer (HNC) cachexia. STUDY DESIGN: Sixty-eight HNC patients were randomly assigned to receive either an Ethanwell/Ethanzyme (EE) regimen enriched with omega-3 fatty acids, micronutrients, and probiotics, or control (Isocal) for a 3-month period. Analysis of covariance was used to examine the association between BW change and variables. RESULTS: Patients with body mass index (BMI) <19 and those receiving the EE regimen consumed fewer daily calories but showed significantly increased BW and maintained higher serum albumin and prealbumin levels than other patients (P<.05). Their BW changes were significantly associated with changes in serum albumin and prealbumin levels. CONCLUSIONS: EE regimen improved BW as well as serum albumin and prealbumin levels in HNC patients with BMI <19.

Distribution of selenium and oxidative stress in breast tumor-bearing mice.

The present study investigated the effects of breast tumors on the blood and tissue distribution of essential trace mineral selenium (Se), and oxidative stress status of mice. Female 10-week-old BALB/cByJNarl mice were randomly assigned into control (CNL) and breast tumor-bearing (TB) groups. TB mice were injected subcutaneously into the right hind thigh with 5 × 10(6) EMT6 mouse mammary tumor cells. After 22 days, we measured Se concentrations, Se-dependent glutathione peroxidase (GPx) activities, and malondialdehyde (MDA) products (indicator of oxidative stress) in plasma, various tissues, and plasma vascular endothelial growth factor (VEGF) concentrations. There were no significant differences in body weights and daily intake between both groups. Compared with the CNL group, TB mice have decreases in plasma Se concentrations and GPx activities, as well as higher plasma VEGF and MDA concentrations. Plasma Se concentrations were also negatively correlated with plasma MDA and VEGF concentrations. Furthermore, tissue Se concentrations and GPx activities in TB animals were lower; whereas the MDA concentrations higher in various tissues including liver, kidney, brain, lung, spleen, and thymic tissues. In conclusion, disruption of Se homeostasis critically reflects oxidative stress in target tissues, thus may increase the risk for progression of breast cancer and metastasis.

Reduction of splenic immunosuppressive cells and enhancement of anti-tumor immunity by synergy of fish oil and selenium yeast.

Growing evidence has shown that regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) abnormally increase in cancer cachectic patients. Suppressions of Tregs and MDSCs may enhance anti-tumor immunity for cancer patients. Fish oil and selenium have been known to have many biological activities such as anti-inflammation and anti-oxidation. Whether fish oil and/or selenium have an additional effect on population of immunosuppressive cells in tumor-bearing hosts remained elusive and controversial. To gain insights into their roles on anti-tumor immunity, we studied the fish oil- and/or selenium-mediated tumor suppression and immunity on lung carcinoma, whereof cachexia develops. Advancement of cachexia in a murine lung cancer model manifested with such indicative symptoms as weight loss, chronic inflammation and disturbed immune functionality. The elevation of Tregs and MDSCs in spleens of tumor-bearing mice was positively correlated with tumor burdens. Consumption of either fish oil or selenium had little or no effect on the levels of Tregs and MDSCs. However, consumption of both fish oil and selenium together presented a synergistic effect--the population of Tregs and MDSCs decreased as opposed to increase of anti-tumor immunity when both fish oil and selenium were supplemented simultaneously, whereby losses of body weight and muscle/fat mass were alleviated significantly.

Role of certain trace minerals in oxidative stress, inflammation, CD4/CD8 lymphocyte ratios and lung function in asthmatic patients.

BACKGROUND: Asthma is associated with increased inflammation, oxidative stress and abnormal immune system function. We determined the distributions of several essential trace minerals and assessed their relationships to factors that are associated with the pathophysiological status of patients with mild/moderate asthma. METHODS: We enrolled 25 asthmatic patients and 25 healthy subjects. We measured: blood trace minerals, zinc (Zn), copper (Cu) and selenium (Se); oxidative stress markers thiobarbituric acid reactive substances (TBARS); antioxidant enzyme activities; percentages of CD4 and CD8 lymphocyte subsets; high-sensitivity C-reactive protein (hs-CRP); and a lung function index (FEV1/FVC%). RESULTS: Compared with healthy subjects, asthmatics had lower concentrations of Zn and Se; higher Cu concentrations, and Cu/Zn and Cu/Se ratios; and lower antioxidant enzyme glutathione peroxidase (GPx), glutathione reductase (GR) and catalase activities. Significantly increased concentrations of hs-CRP, TBARS and CD4/CD8 lymphocyte ratios were also observed. Furthermore, plasma TBARS or hs-CRP concentrations were negatively associated with Se concentrations, but were positively associated with Cu/Se ratios. CD4/CD8 lymphocyte ratios were inversely correlated with Se, while it was positively correlated with Cu/Se ratio. FEV1/FVC% was also significantly correlated with Se concentrations, and Cu/Se and Cu/Zn ratios. CONCLUSIONS: Abnormal distributions of these trace minerals may aggravate oxidative damage and inflammation, increased CD4/CD8 lymphocyte ratios and decreased lung function in asthma.