Assessment of the Proton Pump Inhibitor, Esomeprazole Magnesium Hydrate and Trihydrate, on Pathophysiological Markers of Preeclampsia in Preclinical Human Models of Disease.

Previously, we demonstrated that the proton pump inhibitor, esomeprazole magnesium hydrate (MH), could have potential as a repurposed treatment against preeclampsia, a serious obstetric condition. In this study we investigate the difference in the preclinical effectiveness between 100 µM of esomeprazole MH and its hydration isomer, esomeprazole magnesium trihydrate (MTH). Here, we found that both treatments reduced secretion of sFLT-1 (anti-angiogenic factor) from primary cytotrophoblast, but only esomeprazole MH reduced sFLT-1 secretion from primary human umbilical vein endothelial cells (assessed via ELISA). Both drugs could mitigate expression of the endothelial dysfunction markers, vascular cell adhesion molecule-1 and endothelin-1 (via qPCR). Neither esomeprazole MH nor MTH quenched cytotrophoblast reactive oxygen species production in response to sodium azide (ROS assay). Finally, using wire myography, we demonstrated that both compounds were able to induce vasodilation of human omental arteries at 100 µM. Esomeprazole is safe to use in pregnancy and a candidate treatment for preeclampsia. Using primary human tissues and cells, we validated that esomeprazole is effective in enhancing vascular relaxation, and can reduce key factors associated with preeclampsia, including sFLT-1 and endothelial dysfunction. However, esomeprazole MH was more efficacious than esomeprazole MTH in our in vitro studies.

Plant-derived alkaloid sinomenine potentiates glucocorticoid pharmacodynamics in mitogen-activated human peripheral blood mononuclear cells by regulating the translocation of glucocorticoid receptor.

Sinomenine has been used as an antirheumatic drug in China. Glucocorticoid combined with sinomenine could be an alternative therapeutic approach. In this study, we evaluated the sinomenine potential effect on glucocorticoid pharmacodynamics in vitro using a human peripheral blood mononuclear cell (PBMC) culture system. We also disclosed the possible action mechanism of sinomenine with a focus on P-glycoprotein function and glucocorticoid receptor (GR) translocation into nucleus. The median (range) of methylprednisolone IC50 values against the PBMC proliferation was 3.18 (0.45-6.81) ng/mL, whereas the median (range) IC50 values of methylprednisolone combined with 0.03, 0.3, 3, and 30 µM sinomenine were 1.85 (0.05-5.15), 0.83 (0.10-3.90), 0.56(0.09-1.62), and 0.59(0.05-1.30) ng/mL, respectively. Sinomenine significantly decreased the IC50 values of methylprednisolone and enhanced the immunosuppressive effect of methylprednisolone (p < 0.05). Sinomenine alone regulated the GR translocation in both Jurkat T cells and normal human PBMCs, and the combination of sinomenine and methylprednisolone showed stronger GR-modulatory activity than methylprednisolone alone. Thus, the additive effect of sinomenine to promote the methylprednisolone immunosuppressive efficacy was suggested to be related to nuclear GR-translocation. However, sinomenine did not significantly inhibit the P-glycoprotein function in the activated PBMCs, suggesting that sinomenine's additive effect seemed to be unrelated with the P-glycoprotein inhibition.

Effects of insulin on pharmacodynamics of immunosuppressive drugs against mitogen-activated human peripheral blood mononuclear cells.

CONTEXT: Diabetes mellitus is one of the most common causes of chronic renal failure. Immunosuppressive efficacies of glucocorticoids, calcineurin inhibitors, and mycophenolic acid are possibly affected by insulin after renal transplantation in these patients. OBJECTIVES: We investigated the effects of insulin on responses of mitogen-activated human peripheral blood mononuclear cells (PBMCs) to several immunosuppressive drugs. MATERIALS AND METHODS: Antiproliferative efficacies of prednisolone, hydrocortisone, cyclosporine, tacrolimus, and mycophenolic acid against concanavalin A-stimulated PBMCs were evaluated in the presence of physiological (5 µunits/mL) and super physiological (50 µunits/mL) concentrations of insulin. Insulin-receptor expressions on PBMCs were evaluated by flow cytometry. RESULTS: Insulin itself had no effects on the mitogen-induced proliferation of PBMCs. The IC50 values of cyclosporine against the mitogen-activated PBMCs in the presence of 5 or 50 µunits/mL insulin were significantly higher than those of cyclosporine without insulin (p < 0.05). The IC50 values of mycophenolic acid significantly increased by 50 µunits/mL insulin (p < 0.01). Insulin receptors were detected on the mitogen-activated CD4(+)/CD14(+ )cells in PBMCs. DISCUSSION AND CONCLUSIONS: These results indicate that insulin at even physiological concentration attenuates suppressive efficacies of several immunosuppressive drugs against mitogen-activated proliferation of human PBMCs, possibly via insulin receptors. Insulin used in dialysis patients accompanying diabetes mellitus is suggested to attenuate efficacies of immunosuppressive drugs after renal transplantation.

Immunomodulatory effects of the pentapeptide YGSRS on human peripheral-blood mononuclear cells.

CONTEXT: The pentapeptide YGSRS is originated from coffee bean, while its pharmacological features have little been examined. OBJECTIVES: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+ CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells (PBMCs) activated by T-cell mitogen. MATERIALS AND METHODS: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessed by WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture medium were analyzed with beads-array procedures followed by analysis with flow cytometry. The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. RESULTS: YGSRS at 1-10,000 ng/ml (1.56-15,600 nM) has a tendency to promote the mitogen-activated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affect the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, IL-6 and IL-10 from the activated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in the medium were observed at 1-1000 ng/ml (1.56-1560 nM) (p < 0.05). YGSRS has a tendency to decrease the frequency of Treg cells in the activated PBMCs, but the difference was not statistically significant. DISCUSSION AND CONCLUSIONS: The data suggest that the pentapeptide YGSRS affects the production of several types of cytokines from activated human peripheral T cells, which may modulate Th2 type immunity.

Drug-drug interaction assessment based on a large-scale spontaneous reporting system for hepato- and renal-toxicity, and thrombocytopenia with concomitant low-dose methotrexate and analgesics use.

BACKGROUND: Methotrexate (MTX) is the cornerstone of rheumatoid arthritis (RA) treatment and is highly effective with low-dose intermittent administration. MTX is occasionally used in combination with non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen (APAP)/paracetamol for pain or inflammation control. With MTX treatment, the side effects, such as hepatotoxicity, renal failure, and myelosuppression should be considered. These are also seen with analgesics treatment. METHODS: We used a large spontaneously reported adverse event database (FAERS [JAPIC AERS]) to analyze whether the reporting of adverse events increased upon MTX and analgesic therapy in patients with RA. RESULTS: After identifying RA cases, the crude reporting odds ratios (cRORs) for hepatotoxicity, renal failure, and thrombocytopenia associated with the use of MTX, APAP, or NSAIDs were calculated by disproportionality analysis, which revealed significantly higher cRORs for these events. No analgesics showed consistent positive signals for drug-drug interaction (DDI) with concomitant low-dose MTX analyzed using four algorithms for DDI interaction (the Ω shrinkage measure, additive or multiplicative, and combination risk ratio models). However, in renal failure and thrombocytopenia, loxoprofen (Ω025 = 0.08) and piroxicam (Ω025 = 0.46), and ibuprofen (Ω025 = 0.74) and ketorolac (Ω025 = 3.52), respectively, showed positive signals in the Ω shrinkage measure model, and no consistency was found among adverse events or NSAIDs. CONCLUSIONS: Studies using spontaneous reporting systems have limitations such as reporting bias or lack of patient background; however, the results of our comprehensive analysis support the results of previous clinical or epidemiological studies. This study also demonstrated the usefulness of FAERS for DDI assessment.

Cepharanthine synergistically promotes methylprednisolone pharmacodynamics against human peripheral blood mononuclear cells possibly via regulation of P-glycoprotein/glucocorticoid receptor translocation.

BACKGROUND: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. METHODS: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. RESULTS: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 µM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 µM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 µM of CEP did not influence P-glycoprotein expression. 0.03~0.3 µM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 µM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP.

Polymethoxyflavonoids tangeretin and nobiletin increase glucose uptake in murine adipocytes.

Tangeretin and nobiletin are polymethoxyflavonoids that are contained in citrus fruits. Polymethoxyflavonoids are reported to have several biological functions including anti-inflammatory, anti-atherogenic, or anti-diabetic effects. However, whether polymethoxyflavonoids directly affect glucose uptake in tissues is not well understood. In the current study, we investigated whether tangeretin and nobiletin affect glucose uptake in insulin target cells such as adipocytes. We observed that treatment with tangeretin or nobiletin significantly increased the uptake of [(3) H]-deoxyglucose in differentiated 3T3-F442A adipocytes in a concentration-dependent manner. Data showed that phosphatidyl inositol 3 kinase, Akt1/2, and the protein kinase A pathways were involved in the increase in glucose uptake induced by polymethoxyflavonoids. These data suggest that the anti-diabetic action of polymethoxyflavonoids is partly exerted via these signaling pathways in insulin target tissues.

Methotrexate-related adverse events and impact of concomitant treatment with folic acid and tumor necrosis factor-alpha inhibitors: An assessment using the FDA adverse event reporting system.

Methotrexate (MTX) is an essential anti-rheumatic drug used to treat rheumatoid arthritis (RA). Prevention or management of adverse reactions, including interstitial lung disease (ILD), hepatotoxicity, myelosuppression, and infection, remains fundamental for safe MTX therapy. Using the Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) (JAPIC AERS), we performed disproportionality analyses of adverse events related to MTX use and the impact of concomitant medications. Upon analyzing all reported cases in FAERS between 1997 and 2019, the crude reporting odds ratios (cRORs; 95% confidence intervals) for ILD, hepatotoxicity, myelosuppression, and tuberculosis (TB) in relation to MTX use were 4.00 (3.83-4.17), 1.99 (1.96-2.02), 3.66 (3.58-3.74), and 7.97 (7.65-8.3), respectively. Combining MTX with folic acid (FA) or tumor necrosis factor-alpha inhibitors (TNFis) tended to reduce cRORs for these adverse events (except for TB). Multiple logistic regression analysis in patients with RA was conducted to calculate adjusted reporting odds ratios (aRORs) for age, sex, and MTX treatment patterns (MTX alone and combined with FA and TNFi). Higher age (except for hepatotoxicity) and male sex were significantly associated with adverse events. Combining FA or TNFi with MTX reduced aRORs for MTX-related hepatotoxicity and myelosuppression; in contrast, the effect of FA was not obvious in ILD or TB. Although studies assessing spontaneous reporting systems have limitations such as reporting bias, data from our logistic regression analysis demonstrated that adding FA to MTX-based therapy could help reduce the dose-dependent adverse events of MTX, thereby providing clinical evidence that supports the beneficial effect of FA. This study also demonstrated the usefulness of FAERS in comparing adverse events based on treatment patterns.

Antiproliferative and apoptosis-inducing effects of lipophilic vitamins on human melanoma A375 cells in vitro.

The effects of six lipophilic vitamins: tretinoin (ATRA), vitamin D(3) (VD(3)), VE, VK(1), VK(3), and VK(5) on cell proliferation and apoptosis in human A375 melanoma cells were investigated. VD(3), VK(3), and VK(5) were found to inhibit cell proliferation significantly at concentration ranges of 10-100 µmol/L (p<0.01), while the other vitamins did not show inhibitory effects at 100 µmol/L. VK(3) and VK(5) showed the strongest effects with IC(50) values of less than 10 µmol/L. Dacarbazine slightly inhibited the proliferation of A375 cells at a concentration range of 25-100 µmol/L, but the effects were not statistically significant. VK(3) and VK(5) increased annexin-V positive apoptotic cells, as well as activating caspase-3, in A375 cells. Our findings showed that VD(3), VK(3,) and VK(5) inhibited the growth of dacarbazine resistant human melanoma cells, while ATRA, VE, and VK(1) had little effect on the cell growth. The effects of VK(3) and VK(5) were observed at concentrations lower than 10 µmol/L, which are suggested to have resulted from apoptosis-induction in the melanoma cells.

Development of Japanese Versions of the Control Preferences Scale and Information Needs Questionnaire: Role of Decision-Making and Information Needs for Japanese Breast Cancer Patients.

PURPOSE: The importance of shared decision-making (SDM) between physicians and patients is increasingly recognized. In Japan, patients have shown more willingness to participate in treatment if medical professionals provide sufficient information; however, relationships between physicians and patients have traditionally been asymmetric, with patients accepting information from physicians without discussion. To explore the benefits of SDM in cancer treatment, including confidence in treatment decisions, satisfaction with treatment, and trust in healthcare providers, this study developed Japanese versions of the Control Preference Scale (CPS) and Information Needs Questionnaire (INQ). PATIENTS AND METHODS: Reliability and validity of the CPS and INQ were tested with 49 breast cancer patients. RESULTS: The CPS showed good test-retest reliability (kappa coefficient: 0.61, weighted kappa coefficient: 0.61, Kendall's tau coefficient: 0.61) and acceptable criterion validity. The INQ showed adequate consistency; the mean number of circular triads and coefficient of consistency were 3 (range 0-19) and 0.9 (range 0.37-1), respectively. Using the CPS and INQ to identify patients' roles in decision-making and information needs, results further suggested that breast cancer patients in Japan want to participate in SDM. Medical issues, including disease spread and cure, were found to be of high interest, while social and psychological issues, including sexual attractiveness, genetic risk, and family impact, tended to be low. CONCLUSION: The Japanese CPS and INQ can be used to assess patients' needs to improve care. Further, as patients' information needs change along the care trajectory, these tools should be used throughout treatment.

Molecular mechanisms and therapeutic implications of tetrandrine and cepharanthine in T cell acute lymphoblastic leukemia and autoimmune diseases.

Inappropriately activated T cells mediate autoimmune diseases and T cell acute lymphoblastic leukemia (T-ALL). Glucocorticoid and chemotherapeutic agents have largely extended lives of these patients. However, serious side effects and drug resistance often limit the prognosis of considerable number of the patients. The efficient treatment of autoimmune diseases or T-ALL with drug resistance remains an important unmet demand clinically. Bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine have been applied for the treatment of certain types of autoimmune diseases and cancers, while studies on their action mechanisms and their further applications combined with glucocorticoids or chemotherapeutic agents remains to be expanded. This review introduced molecular mechanisms of tetrandrine and cepharanthine in T cells, including their therapeutic implications. Both tetrandrine and cepharnthine influence the growth of activated T cells via several kinds of signaling pathways, such as NF-κB, caspase cascades, cell cycle, MAPK, and PI3K/Akt/mTOR. According to recent preclinical and clinical studies, P-glycoprotein inhibitory effect of tetrandrine and cepharnthine could play a significant role on T cell-involved refractory diseases. Therefore, tetrandrine or cepharanthine combined with glucocorticoid or other anti-leukemia drugs would bring a new hope for patients with glucocorticoid-resistant autoimmune disease or refractory T-ALL accompanied with functional P-glycoprotein. In conclusion, bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine can regulate several signaling pathways in abnormally activated T cells with low toxicity. Bisbenzylisoquinoline alkaloids deserve to be paid more attention as a lead compound to develop new drugs for the treatment of T cell-involved diseases in the future.

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells.

Sofalcone, 2'-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.

[Corrigendum] Antitumor effects of arsenic disulfide on the viability, migratory ability, apoptosis and autophagy of breast cancer cells.

Following the publication of the above paper, an interested reader drew to our attention the fact that the same ß­actin bands had been included in the western blots featured in Figs. 7 and 10. Upon consulting the authors in relation to this matter, they were able to offer a legitimate explanation for this apparent duplication of the bands; essentially, in the western blotting experiments, different antibodies were used in one membrane to present different target proteins by stripping and reprobing the membrane. That the authors had performed this additional step in their protocol was inadvertently omitted from the 'Western blot analyses' subsection of the Materials and methods section. The authors are grateful to the interested reader for drawing this matter to their attention, and apologize for any inconvenience caused to the readership of the Journal. [the original article was published in Oncology Reports 41: 27-42, 2019; DOI: 10.3892/or.2018.6780].

Tetrandrine enhances glucocorticoid receptor translocation possibly via inhibition of P-glycoprotein in daunorubicin-resistant human T lymphoblastoid leukemia cells.

Glucocorticoids are used as anticancer and immunosuppressive agents, whereas glucocorticoid resistance has been observed in a significant fraction of patients due to overexpression of P-glycoprotein encoded by multi-drug resistance-1 gene. Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from traditional herb Fangji. According to our previous report, tetrandrine potentiated glucocorticoid pharmacodynamics partially via inhibiting P-glycoprotein function. In the present study, we investigated whether glucocorticoid receptor translocation was influenced indirectly by tetrandrine via P-glycoprotein inhibition, using human T lymphoblastoid leukemia MOLT-4 cell line with little P-glycoprotein expression and its multidrug resistant sub-line MOLT-4/DNR exhibiting a large amount of P-glycoprotein. Molecular mechanism investigation suggested that overexpressed P-glycoprotein weakened the glucocorticoid receptor translocation in MOLT-4/DNR cells comparing with the parent MOLT-4 cells. Our data also suggested that tetrandrine enhanced nuclear glucocorticoid receptor translocation in MOLT-4/DNR cells indirectly by dual influences on P-glycoprotein, inhibiting the efflux function and downregulating the protein expression. Therefore, tetrandrine potentiated the cytotoxic effect of methylprednisolone against MOLT-4/DNR cells with less effects on MOLT-4 cells. These effects of tetrandrine were suggested to be beneficial for the treatment of glucocorticoid resistant diseases induced by the overexpression of P-glycoprotein.

Sofalcone, an anti-ulcer chalcone derivative, suppresses inflammatory crosstalk between macrophages and adipocytes and adipocyte differentiation: implication of heme-oxygenase-1 induction.

Sofalcone, 2'-carboxymethoxy-4,4'-bis(3-methyl-2-butenyloxy)chalcone, has been used as an anti-ulcer agent, although its precise molecular mechanism has not been completely understood. In the current study, we tested the effects of sofalcone on the inflammatory crosstalk between macrophages and adipocytes and on the differentiation of pre-adipocytes. We found that sofalcone has a strong suppressive effect on the production of nitric oxide (NO), tumor necrosis factor (TNF)alpha, and monocyte chemoattractant protein (MCP)-1 in the culture medium of a coculture system containing RAW264.7 macrophages and 3T3-F442A adipocytes stimulated with lipopolysaccharide (LPS). The suppressive effect of sofalcone on NO production was attenuated by treatment with tin-protoporphyrin (SnPP), a heme-oxygenase (HO)-1 inhibitor. Western blotting analysis showed that sofalcone increased HO-1 expression in both 3T3-F442A mature adipocytes and undifferentiated fibroblasts. Sofalcone also inhibited the differentiation of 3T3-F442A pre-adipocytes into adipocytes, which was restored by SnPP treatment. These results suggest that sofalcone has preferable properties for obesity or metabolic syndrome.

Bevacizumab Versus Anti-preeclamptic Drugs: Evaluation With Three-dimensionally Co-cultured Human Mini Tumors.

BACKGROUND/AIM: Both bevacizumab (BEV) and soluble fms-like tyrosine kinase-1 (sFlt-1) have demonstrated anti-angiogenic effects, thereby causing hypertension and proteinuria. We hypothesized that anti-preeclamptic drugs that combat the action of sFlt-1 may reduce BEV's anti-tumor efficacy. MATERIALS AND METHODS: 3D co-cultured human mini-tumors consisting of endothelial cells, fibroblasts, and cancer cells were developed. The influence of anti-preeclamptic drugs and BEV on the invasion of mini-tumors embedded in collagen gel was evaluated. RESULTS: Mini-tumor spheroids that contained MDA-MB-231 cells showed higher invasion ability than spheroids with A549. Among the six anti-preeclamptic drugs investigated, only nicorandil enhanced the invasion of mini-tumors and inhibited the action of BEV. Glibenclamide, an ATP-sensitive potassium channel inhibitor, completely quenched the action of nicorandil on mini-tumors. CONCLUSION: In the human mini-tumor model, nicorandil aggravated the invasion of mini-tumors. These data raise the possibility that concomitant use of nicorandil counteracts the efficacy of BEV therapy.

Antitumor effects of arsenic disulfide on the viability, migratory ability, apoptosis and autophagy of breast cancer cells.

In the present study, the antitumor effects of arsenic disulfide (As2S2) on the proliferative, survival and migratory ability of human breast cancer MCF­7 and MDA­MB­231 cells were investigated, and its potential underlying molecular mechanisms with an emphasis on cell cycle arrest, apoptosis induction, autophagy induction and reactive oxygen species (ROS) generation were determined. The results indicated that As2S2 significantly inhibited the viability, survival and migration of breast cancer cells in a dose­dependent manner. In addition, it was identified that As2S2 induced cell cycle arrest primarily at G2/M phase in the two breast cancer cell lines by regulating the expression of associated proteins, including cyclin B1 and cell division cycle protein 2. In addition to cell cycle arrest, As2S2 also triggered the induction of apoptosis in cells by activating the expression of pro­apoptotic proteins, including caspase­7 and ­8, as well as increasing the B­cell lymphoma 2 (Bcl­2)­associated X protein/Bcl­2 ratio, while decreasing the protein expression of anti­apoptotic B­cell lymphoma extra­large. In addition, As2S2 stimulated the accumulation of microtubule­associated protein 1A/1B­light chain 3 (LC3)­II and increased the LC3­II/LC3­I ratio, indicating the occurrence of autophagy. As2S2 treatment also inhibited the protein expression of matrix metalloproteinase­9 (MMP­9), but increased the intracellular accumulation of ROS in the two breast cancer cell lines, which may assist in alleviating metastasis and attenuating the progression of breast cancer. Taken together, the results of the present study suggest that As2S2 inhibits the progression of human breast cancer cells through the regulation of cell cycle arrest, intrinsic and extrinsic apoptosis, autophagy, MMP­9 signaling and ROS generation.

Arsenic Disulfide Combined with L-Buthionine-(S, R)-Sulfoximine Induces Synergistic Antitumor Effects in Two-Dimensional and Three-Dimensional Models of MCF-7 Breast Carcinoma Cells.

Three-dimensionally (3D) cultured tumor cells (spheroids) exhibit more resistance to therapeutic agents than the cells cultured in traditional two-dimensional (2D) system (monolayers). We previously demonstrated that arsenic disulfide (As2S2) exerted significant anticancer efficacies in both 2D- and 3D-cultured MCF-7 cells, whereas 3D spheroids were shown to be resistant to the As2S2 treatment. L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been regarded to be a potent candidate for combinatorial treatment due to its GSH modulation function. In the present study, we introduced BSO in combination with As2S2 at a low concentration to investigate the possible enhancing anticancer efficacy by the combinatorial treatment on 2D- and 3D-cultured MCF-7 cells. Our results presented for the first time that the combination of As2S2 and BSO exerted potent anticancer synergism in both MCF-7 monolayers and spheroids. The IC50 values of As2S2 in combinatorial treatment were significantly lower than those in treatment of As2S2 alone in both 2D- and 3D-cultured MCF-7 cells (P<0.01, respectively). In addition, augmented induction of apoptosis and enhanced cell cycle arrest along with the regulation of apoptosis- and cell cycle-related proteins, as well as synergistic inhibitions of PI3K/Akt signals, were also observed following co-treatment of As2S2 and BSO. Notably, the combinatorial treatment significantly decreased the cellular GSH levels in both 2D- and 3D-cultured MCF-7 cells in comparison with each agent alone (P<0.05 in each). Our results suggest that the combinatorial treatment with As2S2 and BSO could be a promising novel strategy to reverse arsenic resistance in human breast cancer.

Tetrandrine and cepharanthine induce apoptosis through caspase cascade regulation, cell cycle arrest, MAPK activation and PI3K/Akt/mTOR signal modification in glucocorticoid resistant human leukemia Jurkat T cells.

Tetrandrine (TET) and cepharanthine (CEP) are two bisbenzylisoquinoline alkaloids isolated from the traditional herbs. Recent molecular investigations firmly supported that TET or CEP would be a potential candidate for cancer chemotherapy. Prognosis of patients with glucocorticoid resistant T cell acute lymphoblastic leukemia (T-ALL) remains poor; here we examined the anti-T-ALL effects of TET and CEP and the underlying mechanism by using the glucocorticoid resistant human leukemia Jurkat T cell line in vitro. TET and CEP significantly inhibited cell viabilities and induced apoptosis in dose- and time-dependent manner. Further investigations showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9, but also increased the expression of effector caspases such as caspase-3 and 6. As the important markers of apoptosis, p53 and Bax were both upregulated by the treatment of TET and CEP. However, TET and CEP paradoxically increased the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, and activated the survival protein NF-κB, leading to high expression of p-NF-κB. Cell cycle arrest at S phase accompanied by increase in the amounts of cyclin A2 and cyclin B1, and decrease in cylcin D1 amount in cells treated with TET or CEP will be another possible mechanism. During the process of apoptosis in Jurkat T cells, treatment with TET or CEP also increased the phosphorylation of JNK and p38. The PI3K/Akt/mTOR signaling pathway modification appears to play significant role in the Jurkat T cell apoptosis induced by TET or CEP. Moreover, TET and CEP seemed to downregulate the expressions of p-PI3K and mTOR in an independent way from Akt, since these two drugs strongly stimulated the p-Akt expression. These results provide fundamental insights into the clinical application of TET or CEP for the treatment of patients with relapsed T-ALL.

Suppression of blood lipid concentrations by volatile Maillard reaction products.

OBJECTIVE: Although the chemistry of Maillard reaction products (MRPs) in foods has been well studied, few reports on the nutritional characteristics of MRPs in experimental animals and humans have been found. In this study, our interest was focused on the volatile MRPs (vMRPs) found in heated foods. METHODS: To confirm the metabolic oxidations of six methylpyrazines and pyrrole-2-carboxaldehyde to carboxylic acid derivatives in vivo, we administrated these compounds orally to Wistar rats with a single dose of 50 mg/kg. Urine samples were collected over 24 h, followed by determination using high-performance liquid chromatographic procedures. Eight pyrazinoic acids, 2-furoic acid, and 5-hydroxymethyl-2-furoic acid were administered orally to rats with a single dose of 100 or 300 mg/kg, and blood non-esterified fatty acid and triacylglycerol concentrations were analyzed. RESULTS: Monomethylpyrazine, 2,3-, 2,5-, and 2,6-dimethylpyrazine, trimethylpyrazine, and tetramethylpyrazine were metabolized to a corresponding pyrazinoic acid such as non-substituted pyrazinioic acid, 3-, 5-, or 6-methylpyrazinoic acid, 3,5-, 3,6-, and 5,6-dimethylpyrazinoic acid, and trimethylpyrazinoic acid, in appropriate yields, respectively. Further, pyrrole-2-carboxaldehyde was metabolized to pyrrole-2-carboxylic acid. Non-substituted and 5-methylpyrazinoic acid and 2-furoic and 5-hydroxymethyl-2-furoic acid showed significant non-esterified fatty acid-lowering effects. 5-Methyl and 6-methylpyrazinoic acid and 2-furoic acid showed significant triacylglycerol-lowering effects. Pyrazinoic acids with methyl substitution at position 3 showed no lipid-lowering effect. CONCLUSION: These results suggest that the vMRPs such as methylpyrazines are metabolized to their corresponding pyrazinoic acids. These vMRPs and their metabolites exhibit blood lipid-lowering effects in rats.