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.

Individual Lymphocyte Sensitivity to Steroids as a Reliable Biomarker for Clinical Outcome after Steroid Withdrawal in Japanese Renal Transplantation.

Recently, steroid reduction/withdrawal regimens have been attempted to minimize the side effects of steroids in renal transplantation. However, some recipients have experienced an increase/resumption of steroid administrations and acute graft rejection (AR). Therefore, we investigated the relationship between the individual lymphocyte sensitivity to steroids and the clinical outcome after steroid reduction/withdrawal. We cultured peripheral blood mononuclear cells (PBMCs) isolated from 24 recipients with concanavalin A (Con A) in the presence of methylprednisolone (MPSL) or cortisol (COR) for four days, and the 50% of PBMC proliferation (IC50) values and the PBMC sensitivity to steroids were calculated. Regarding the experience of steroid increase/resumption and incidence of AR within one year of steroid reduction/withdrawal, the IC50 values of these drugs before transplantation in the clinical event group were significantly higher than those in the event-free group. The cumulative incidence of steroid increase/resumption and AR in the PBMC high-sensitivity groups to these drugs before transplantation were significantly lower than those in the low-sensitivity groups. These observations suggested that an individual's lymphocyte sensitivity to steroids could be a reliable biomarker to predict the clinical outcome after steroid reduction/withdrawal and to select the patients whose dose of steroids can be decreased and/or withdrawn after transplantation.

Effects of vitamin K2 combined with methotrexate against mitogen-activated peripheral blood mononuclear cells of healthy subjects and rheumatoid arthritis patients.

BACKGROUND: Methotrexate (MTX) is used as anchor drug for patients with early and established rheumatoid arthritis (RA). Vitamin K2 administration was also reported to be associated with decreased disease activity in RA. OBJECTIVES: Immunosuppressive pharmacodynamics of vitamin K2 combined with MTX was investigated. METHODS: Mitogen-activated peripheral blood mononuclear cells (PBMCs) were used to evaluate immunosuppressive pharmacodynamics of drugs in vitro. RESULTS: Vitamin K2 alone dose-dependently suppressed T cell mitogen-activated proliferation of PBMCs of both healthy subjects and RA patients. 446.5 and 2232.5 ng/mL vitamin K2 significantly decreased the IC50 values of MTX on the proliferation of PBMCs of RA patients, with little influences on the pharmacodynamics of MTX in the healthy PBMCs. 4465 ng/mL vitamin K2 potentiated the pharmacodynamics of MTX in both RA patients and healthy PBMCs. The additional effects of vitamin K2 to potentiate the suppressive effects of MTX seemed not to be related to the regulation of CD4+ CD25+ T cells or CD4+ CD25+ Foxp3+ Treg cells. MTX alone at 100 ng/mL significantly decreased the percentage of CD4+ T cells in PBMCs of healthy subjects (p < 0.001) with a slight influence in that of RA patients (not significant) and the combination did not show synergistic inhibitory effect. Vitamin K2 alone tended to suppress the secretion of IL-17, IFN-γ, and TNF-α from the activated PBMCs of RA patients with smaller influences on the cytokine productions from healthy PBMCs. These additional effects of vitamin K2 were also observed in combination with MTX. CONCLUSION: The above information may partially elucidate the potentiation effects of vitamin K2 on the immunosuppressive efficacy of MTX.

Pharmacodynamic Drug-Drug Interaction on Human Peripheral Blood Mononuclear Cells Between Everolimus and Tacrolimus at the Therapeutic Concentration Range in Renal Transplantation.

BACKGROUND Everolimus (EVL) plus tacrolimus (TAC) therapy is effective and safe in renal transplantation. However, the pharmacokinetic and pharmacodynamic information for EVL combined with TAC is limited. We investigated the pharmacodynamic drug-drug interaction between EVL and TAC at their therapeutic concentration range. MATERIAL AND METHODS Isolated peripheral blood mononuclear cells (PBMCs) from 22 healthy participants aged 22 to 24 years were cultured with concanavalin A (Con A) in the presence of EVL and/or TAC for 4 days, and the proliferation rate of the PBMCs was calculated. RESULTS TAC promoted the inhibitory efficacy of EVL against the mitogen-activated proliferation of PBMCs at the EVL therapeutic concentration range. When 0.175 ng/mL or more of TAC was combined with 30 ng/mL or more of EVL, the antagonistic effect of TAC on the inhibitory efficacy of EVL against the mitogen-activated proliferation of PBMCs was observed. Conversely, when 0.4 ng/mL TAC and 10 ng/mL or more of EVL were combined, the antagonistic effect of EVL on the inhibitory efficacy of TAC against the mitogen-activated proliferation of PBMCs was observed. CONCLUSIONS The pharmacodynamic synergistic efficacy of EVL and TAC in combination on mitogen-activated PBMCs was evident at the therapeutic concentration range, which is used in renal transplantation. However, these drugs antagonize each other to suppress the proliferation of activated PBMCs at concentrations higher than those clinically used.

Vitamin K2 Suppresses Proliferation and Inflammatory Cytokine Production in Mitogen-Activated Lymphocytes of Atopic Dermatitis Patients through the Inhibition of Mitogen-Activated Protein Kinases.

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10-100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10-100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

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.

Absolute configuration of tetrandrine and isotetrandrine influences their anti-proliferation effects in human T cells via different regulation of NF-κB.

Natural compound tetrandrine was reported to inhibit the proliferation of T cells by inhibiting activation of NF-κB. Chemically, isotetrandrine differs from tetrandrine only in the stereochemistry at the chiral centers. The present study aimed to compare their anti-proliferation effects on human T cells with a focus on NF-κB. The IC50 values of tetrandrine against MOLT-4 cells, MOLT-4/DNR cells, and concanavalin A-activated peripheral blood mononuclear cells of healthy subjects and dialysis patients were 4.43 ± 0.22, 3.62 ± 0.22, 1.91 ± 0.22 and 3.03 ± 0.28 µM, respectively. Whereas, the IC50 values of isotetrandrine against the above immune cells were 2.19 ± 0.27, 2.28 ± 0.33, 1.29 ± 0.14 and 1.55 ± 0.26 µM, respectively. The inhibitory effect of isotetrandrine against the proliferation of T cells was stronger than that of tetrandrine significantly (p < 0.05). Molecular mechanism investigation showed that 10 µM of isotetrandrine largely decreased the expression of p-NF-κB and NF-κB in both MOLT-4 and MOLT-4/DNR T cells (p < 0.05), whereas 10 µM of tetrandrine slightly inhibited the phosphorylation of p-NF-κB with little influence on the expression of NF-κB. Taken together, absolute configurations of tetrandrine and isotetrandrine are suggested to influence on their anti-proliferation effects in human T cells via different regulation of NF-κB.

Effects of sinomenine on the proliferation, cytokine production, and regulatory T-cell frequency in peripheral blood mononuclear cells of rheumatoid arthritis patients.

Sinomenine (SN) is a plant-derived alkaloid isolated from Caulis Sinomenii. It has been approved by the State Food and Drug Administration of China for treating rheumatoid arthritis (RA) nearly 20 years ago. To investigate the anti-RA mechanism of SN, a lot of scholars reported the immunosuppressive effect of SN on T lymphocytes. We continued to evaluate the suppressive function of SN by using human peripheral blood mononuclear cells (PBMCs) isolated from RA patients. As the positive control, 10 ng/ml of methylprednisolone (MP) showed the antiproliferation effect on mitogen-activated PBMCs of RA patients significantly (*p < .05). Meanwhile, MP decreased the frequency of CD4+ CD25+ T cells and suppressed the secretion of inflammatory Th1/Th2/Th17 cytokines such as IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α. However, SN at concentrations of 0.3-30 µM, showed little suppressive effects on the proliferation of PBMCs of RA patients. We did not observe any suppressive effects of SN on percentages of CD4+ T cells and CD4+ CD25+ T cells in the mitogen-activated PBMCs of RA patients. The influence of SN on the percentage of CD4+ CD25+ Foxp3+ T cells was also limited. Finally, even 30 µM of SN did not influence the secretion of Th1/Th2/Th17 cytokine significantly. The present study provided evidence that anti-RA mechanism of SN seems not to be related with the suppressive effects on peripheral T cells.

Suppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells of rheumatoid arthritis patients.

OBJECTIVE: To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. MATERIALS AND METHODS: Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. RESULTS: Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47 ± 4,910.02 ng/mL (mean ± SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p < 0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). It would be possible to predict the pharmacodynamics of vitamin K2 in RA patients according to the above factors. Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83 ± 12.47 ng/mL (mean ± SD). IC50 values of methotrexate only showed significant correlation with ACPA (p = 0.0158, r = 0.6905), which suggests that ACPA might be a suitable predictor of the pharmacodynamics of methotrexate. CONCLUSION: The above information suggests that vitamin K2 could provide a benefit for the treatment of RA patients via its immunosuppressive function.

Potent antitumor activity of cepharanthine against triple-negative breast cancer spheroids compared with tetrandrine.

Cepharanthine (CEP) is a bis-bynzelisoquinoline alkaloid from the same class as the anticancer agent tetrandrine (TET). However, the effects of CEP against breast cancer have not been extensively studied, despite its long therapeutic history with low toxicity against other types of cancer. 3D culture systems more accurately mimic the human body and address the limitations of determining drug effectiveness compared with 2D culture systems. In the present study, the antitumor activities of TET and CEP were compared in 3D culture systems in triple-negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor-positive breast cancer MCF-7 cell lines. Cell viability, apoptosis and cytotoxicity assays were performed to determine the total number of live or dead cells, the IC50 values, the number of apoptotic cells and spheroid roundness. Viability suppression of MDA-MB-231 cells was significantly greater with both TET and CEP compared with that of MCF-7 cells, and the roundness of MDA-MB-231 spheroids treated with CEP was decreased significantly compared with that of spheroid treated with TET. Cytoplasmic shrinkage in each cell line significantly increased with the treatment of TET compared with the control; however, this effect was stronger with CEP. The ratio of dead/live cells in each cell line treated with TET and CEP increased in a dose-dependent manner. Overall, the present study demonstrated that CEP had greater cell toxicity in 3D spheroids of breast cancer cells compared with TET, suggesting that CEP may have a stronger antitumor activity on TNBC spheroids compared with TET.

JNK and Autophagy Independently Contributed to Cytotoxicity of Arsenite combined With Tetrandrine via Modulating Cell Cycle Progression in Human Breast Cancer Cells.

Novel therapeutic strategies for breast cancer are urgently needed due to the sustained development of drug resistance and tumor recurrence. Trivalent arsenic derivative (arsenite, AsIII) has been reported to induce cytotoxicity in breast cancer cells. We recently demonstrated that AsIII plus tetrandrine (Tetra), a Chinese plant-derived alkaloid, exerted potent antitumor activity against human breast cancer cells, however, the underlying mechanisms for their action have not been well defined. In order to provide fundamental insights for understanding the action of AsIII plus Tetra, the effects of the combined regimen on two breast cancer cell lines T47D and MDA-MB-231 were evaluated. Compared to T47D cells, MDA-MB-231 cells were much more susceptible to the synergistic cytotoxic effects of AsIII and Tetra. Besides the induction of apoptotic/necrotic cell death, S-phase arrest and autophagic cell death were also observed in MDA-MB-231 cells. Exposure of MDA-MB-231 cells to AsIII and Tetra caused the activation of MAPKs. Cytotoxicity of the combined regimen in MDA-MB-231 cell was significantly abrogated by SP600125, a potent c-Jun N-terminal kinase (JNK) inhibitor. However, similar abrogation was not caused by p38 and ERK inhibitors. The addition of either autophagy inhibitors (3-methyladenine or wortmannin) or SP600125 corrected the combined regimen-triggered S-phase arrest, whereas had little effect on the apoptosis/necrosis induction in the cells. Surprisingly, SP600125NC, a negative control for SP600125, significantly strengthened S-phase arrest and the cytotoxicity induced by the combined regimen. The addition of SP600125 did not alter autophagy induction. In conclusion, the cytotoxicity of AsIII combined with Tetra was attributed to the induction of S-phase arrest, apoptotic/necrotic and autophagic cell death. The enhanced cytotoxicity of the two drugs by SP600125NC might be explained by its capability to strengthen S-phase arrest. Our results suggested that JNK and autophagy independently contributed to the cytotoxicity via modulating cell cycle progression. The study further provides fundamental insights for the development of AsIII in combination with Tetra for patients with different types of breast cancer.

Methylprednisolone potentiates tetrandrine pharmacodynamics against human T lymphoblastoid leukemia MOLT-4 cells via regulation of NF-κB activation and cell cycle transition.

Low response to glucocorticoid (GC) predicts therapeutic failure in acute T lymphoblastic leukemia (T-ALL). The efficient and safe strategies are still required for the treatment of relapsed T-ALL. Our previous study revealed that tetrandrine induces apoptosis in human T lymphoblastoid leukemia cells possibly via activation of NF-κB. GCs are recognized as typical NF-κB inhibitors and are used for the treatment of T-ALL patients. In the present study, we examined whether methylprednisolone (MP) potentiates the cytotoxic effect of tetrandrine (TET) via NF-κB regulation by using human T lymphoblastoid leukemia MOLT-4 cells. WST-8 assay data showed that nM grade of MP increased cytotoxicity of TET against MOLT-4 cells in vitro. This effect seemed to be related to the potentiation of TET action by MP to induce apoptosis. Meanwhile, the combination also impeded the transition of cell cycle from G0/G1 phase to S phase. However, the regulation effect of this combination on cell cycle had no relationship with cyclin signaling pathway, since the drug-combination did not influence on the expression of cyclin A2/B1/D1 in MOLT-4 cells. On the other hand, the combination significantly inhibited the phosphorylation of NF-κB (p < 0.01). These results suggest that nM grade of MP potentiates the cytotoxic effect of TET possibly via regulation of NF-κB activation and "G0/G1 to S" phase transition in human T lymphoblastoid leukemia MOLT-4 cells. Combination of TET and MP may provide a new therapeutic strategy for relapsed T-ALL.

Bisbenzylisoquinoline alkaloids and P-glycoprotein function: A structure activity relationship study.

Conflicts with the notion that specific substrate interactions were required in the control of reaction path in active transport systems, P-glycoprotein showed extraordinarily low specificity. Therefore, overexpression P-glycoprotein excluded a large number of anticancer agents from cancer cells, and multidrug resistance happened. Several kinds of bisbenzylisoqunoline alkaloids were reported to modulate P-glycoprotein function and reverse drug resistance. In order to provide more information for their structure activity relationship on P-glycoprotein function, the effects of tetrandrine, isotetrandrine, fangchinoline, berbamine, dauricine, cepharanthine and armepavine on the P-glycoprotein function were compared by using daunorubicin-resistant leukemia MOLT-4 cells in the present study. Among them, tetrandrine exhibited the strongest P-glycoprotein inhibitory effect, followed with fangchinoline and cepharanthine, and subsequently with berbamine or isotetrandrine. However, dauricine and armepavine showed little influence on the P-glycoprotein function. These data revealed that the 18-membered ring of the bisbenzylisoquinoline alkaloids maintained the P-glycoprotein inhibitory activity, suggesting that double isoquinoline units connected by two oxygen bridges were indispensable. Moreover, stereo-configuration of bisbenzylisoquinoline 3D structures determined their inhibitory activities, which provided a new viewpoint to recognize the specificity of binding pocket in P-glycoprotein. Our data also indicated that 3D chemical structure was more sensitive than 2D to predict the P-glycoprotein inhibitory-potencies of bisbenzylisoqunoline alkaloids.

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.

[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].

Midazolam Intoxication in a Premature Neonate.

PURPOSE: We report the case of a male neonate with a respiratory disorder who developed adverse cardiorespiratory symptoms after the continuous infusion of midazolam. METHODS: To clarify the cause of cardiogenic shock, we performed whole exome sequencing and screened relative single-nucleotide variants of 2 cytochrome P450 (CYP) isoforms, CYP3A4 and CYP3A5, which play a dominant role in the metabolic elimination of midazolam. We measured endogenous cortisol 6ß-hydroxylation clearance to phenotypically assess CYP3A activity. FINDINGS: The CYP3A activity level in the patient was significantly lower than the mean CYP3A activity level in healthy adults. Three intronic mutations in the CYP3A4 and CYP3A5 isoforms were detected in the patient. IMPLICATIONS: Our findings suggest that the midazolam concentration in plasma was achieved at above the steady-state concentration during continuous infusion used to sedate neonates receiving mechanical ventilatory support. Evaluation of the drug-metabolizing ability based on CYP3A might be useful if adverse electrophysiologic variables or the induction of tachycardia occur because of delayed elimination.

Cytotoxic effects of vitamins K1, K2, and K3 against human T lymphoblastoid leukemia cells through apoptosis induction and cell cycle arrest.

The present study was undertaken to evaluate cytotoxic effects of vitamin K1 (phylloquinone), vitamin K2 (menaquinones), and vitamin K3 (menadione) against human T lymphoblastoid leukemia cells, Jurkat T cells, MOLT-4 cells, and P-glycoprotein-expressing multidrug-resistant MOLT-4/DNR cells. Vitamins K2 and K3, but not vitamin K1, reduced viabilities of Jurkat, MOLT-4, and MOLT-4/DNR cells. The influence potency of vitamin K3 was larger than that of vitamin K2 in all of the three cell lines. MOLT-4/DNR cells seemed to be more sensitive toward the effects of vitamins K2 and K3. The cytotoxicity of vitamins K2 and K3 on these leukemia cells seems to be related to apoptosis induction and cell cycle arrest. Vitamin K2 and K3 treatment induced cleavage of PARP obviously. Moreover, vitamins K2 and K3 specifically down-regulated the expressions of cyclin A2 in all of the three cell lines. However, the effects of vitamins K2 and K3 on the cell cycle profiling in Jurkat, MOLT-4, and MOLT-4/DNR cells varied with the cell type. Vitamins K2 and K3 also decreased the viability of mitogen-activated human peripheral blood mononuclear cells. Our observations suggest that vitamins K2 and K3 have bilateral cytotoxic effects on activated human peripheral lymphocytes and the human leukemic T cells.

Impact of Machine Perfusion on Sinusoid Microcirculation of Liver Graft Donated After Cardiac Death.

BACKGROUND: The present study examined the impact of oxygenated machine perfusion on preservation of liver grafts donated after cardiac death by measuring sinusoidal endothelial injury and microcirculatory disturbances. MATERIALS AND METHODS: Fifteen porcine livers were retrieved 60 min after warm ischemia and allocated into three groups as follows: (1) CS group: static cold storage, (2) HMP group: oxygenated hypothermic perfusion preservation, (3) SNMP group: oxygenated subnormothermic perfusion preservation. The liver grafts donated after cardiac death were preserved for 4 h in different treatment conditions mentioned previously, then subject to ex vivo reperfusion for 2 h using diluted allogeneic blood. The hemodynamic parameters, liver function tests, tissue adenosine triphosphate (ATP) levels, and immunohistochemical findings were investigated. RESULTS: The number of sinusoidal epithelial cells and trabecular structures were maintained after 4 h of preservation in the CS, HMP, and SNMP group. Liver tissue ATP levels after 4 h of preservation in the HMP and SNMP groups were significantly higher compared with that in the CS group. The sinusoidal epithelial cells were significantly exfoliated to a more severe extent in the CS group than in the HMP and SNMP groups. Intrasinusoidal platelet aggregation occurred more frequently in the CS group than in the HMP and SNMP groups. CONCLUSIONS: The results indicated that oxygenated machine perfusion preservation was important to prevent the depletion of tissue ATP and maintain sinusoidal homeostasis regardless of the perfusate temperature. Our findings suggest oxygenated machine perfusion preservation as an effective alternative to static cold storage.

Initial perfusate purification during subnormothermic machine perfusion for porcine liver donated after cardiac death.

Improvement of machine perfusion (MP) technologies is required to enhance organ quality for donor after cardiac death (DCD) grafts. Installing a dialyzer or a filter into the perfusion circuit to maintain the perfusate condition has some advantages. However, the consequences of purification perfusate during subnormothermic machine perfusion (SNMP) remain unexplained. In this study, the effects of initial purification perfusate with simple method of replacing the first 0.5-L perfusate during SNMP were investigated to consider installation effect of the filter or the dialyzer. Porcine liver grafts, which have 60-min warm ischemia time, were procured to imitate the DCD graft condition. Purified SNMP (PSNMP) results were compared with simple cold storage and conventional SNMP. In PSNMP, initial perfusate of 0.5 L was removed to substitute for purification. After preservation process, the preserved grafts were reperfused with diluted autologous blood for 2 h under normothermic machine perfusion condition to evaluate the liver function using an isolated reperfusion model. The vascular pressures, enzyme release rates and the metabolic indexes during reperfusion were analyzed. The pressures in the hepatic artery after reperfusion 60 min were significantly lower in PSNMP group compared with cold storage (CS) and SNMP groups. In addition, lactate dehydrogenase and alkaline phosphatase were significantly lower after PSNMP than after the CS or SNMP. Also, the metabolic indexes of hyaluronic acid and lactate were significantly decreased by purifying the perfusate in MP preservation than in CS or SNMP. The effectiveness of initial purification perfusate during SNMP was investigated.

Vitamin K2 immunosuppressive effect on pediatric patients with atopic dermatitis.

BACKGROUND: Over 20 kinds of steroids, tacrolimus ointments, and cyclosporine capsules are usually recommended for the treatment of atopic dermatitis (AD), depending on the symptoms of patients. However, several side effects sometimes occur with the extensive use of these agents for the treatment of pediatric AD patients. The purpose of this study was to explore whether vitamin K2 could be a new immunosuppressive candidate for pediatric patients with AD. METHODS: The immunosuppressive efficacy of vitamin K2 was evaluated through a cell-culture procedure using mitogen-activated peripheral blood mononuclear cells (PBMCs) obtained from pediatric AD patients. RESULTS: The mean (SD) IC50 value of vitamin K2 for the proliferation of concanavalin A-activated PBMCs was 15.37 (30.05) µmol/L, while the value for tacrolimus was 0.10 (0.28) ng/mL (0.12 (0.35) nmol/L). There was a significant correlation between the IC50 values for vitamin K2 and those for tacrolimus (P = 0.0001, r = 0.8871). However, there was no significant correlation between the IC50 values of vitamin K2 and those of cyclosporine A or methylprednisolone. A significant correlation between the IC50 values of vitamin K2 or tacrolimus and blood eosinophil counts (P = 0.0099, r = 0.7086 and P = 0.0032, r = 0.7722, respectively) was observed. CONCLUSION: Vitamin K2 -inhibited T-cell mitogen stimulated proliferation of PBMCs from pediatric AD patients in a dose-dependent manner. The PBMCs from pediatric AD patients were more sensitive to the immunosuppressive efficacy of vitamin K2 than the PBMCs from healthy subjects. The individual immunosuppressive pharmacological efficacy of vitamin K2 and of tacrolimus could be inferred from the blood eosinophil count of pediatric AD patients.