Baodong Ye

Total coumarins of Hedyotis diffusa induces apoptosis of myelodysplastic syndrome SKM-1 cells by activation of caspases and inhibition of PI3K/Akt pathway proteins

ETHNOPHARMACOLOGICAL RELEVANCE Hedyotis diffusa is an ethno-medicine used for anti-cancer treatment in the clinic of traditional Chinese medicine (TCM). The total coumarins of Hedyotis diffusa (TCHD) was a selected extract with observed antiproliferative activity, which has not been tested in treatment of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). AIM OF THE STUDY This study aimed to evaluate the apoptosis-inducing effect of TCHD on human MDS cell line (SKM-1) and explore its action mechanism in association with caspase family and PI3K/Akt signaling pathway. MATERIALS AND METHODS The chemical constituents and total coumarins content of TCHD were determined by High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) and UV-vis spectrophotometry, respectively. MTT assay, Hoechst 33258 staining, and Annexin V-FITC/PI double labeling were applied to evaluate TCHDs efficacy on SKM-1 cells. Western blot analysis was also used to clarify the action mechanism of TCHD on protein expression level. RESULTS Two compounds, p-coumaric acid and E-6-O-p-coumaroyl scandoside methyl ester, were identified in TCHD, and its total coumarins content reached 87.4%. By MTT assay, apoptosis-inducing effect of TCHD on SKM-1 cells was found in a dose-dependent manner after 24-48h treatment, with IC50 values of 104.48μg/ml and 100.66μg/ml, respectively. Morphological and flow cytometry observation also confirmed such effect of TCHD. Western blot analysis clarified its action mechanism associating with the activation of caspases and inhibition of PI3K/Akt pathway proteins. CONCLUSIONS This is the first report regarding the apoptosis-inducing efficacy and mechanism of TCHD on SKM-1 cells, providing a promising candidate of TCM for MDS and AML therapy with fewer side effects.

Arsenic trioxide-enhanced, matrine-induced apoptosis in multiple myeloma cell lines.

Matrine and arsenic trioxide are monomers used in traditional Chinese medicine possessing anti-myeloma activities. In this study, we evaluated the effects and mechanisms of matrine, arsenic trioxide, and their combination therapy on the proliferation and apoptosis of the myeloma cell lines RPMI8226 and U266. The effects of growth inhibition were measured by MTT, and apoptotic cells were analyzed by Hoechst 33258 staining and flow cytometry. The levels of caspase-3, poly (ADP-ribose) polymerase (a DNA repair enzyme), Bcl-2 and survivin (antiapoptotic signaling proteins), Bim (a proapoptotic signaling protein), total AKT, and phosphorylated AKT were evaluated by Western blot. Matrine significantly inhibited proliferation of RPMI8226 and U266 cell lines in a dose- and time-dependent manner with an IC₅₀ at 24 h of 2.25 g/L and 2.18 g/L, and at 48 h of 1.64 g/L and 1.58 g/L, respectively. Arsenic trioxide also displayed a dose- and time-dependent inhibition of growth of multiple myeloma cell lines, and synergistic effects occurred when the two were combined. Matrine (0.5, 1.0 g/L) and arsenic trioxide (2, 4 ug/mL) induced the apoptosis of myeloma cells; more early-stage apoptotic cells were seen with the combination therapy (matrine 0.5 g/L plus arsenic trioxide 2 ug/mL and matrine 1.0 g/L plus arsenic trioxide 4 ug/mL). Activation of caspase-3 and poly (ADP-ribose) polymerase, upregulation of Bim expression, downregulation of Bcl-2, survivin expression, as well as inhibition of phosphorylated AKT related to matrine (0, 0.25, 0.5, 1.0, and 2.0 g/L)-mediated apoptosis, and the effects were enhanced when arsenic trioxide (8 ug/mL) was combined with matrine (1.0 g/L). In conclusion, matrine displayed anti-myeloma effects through apoptotic induction, and arsenic trioxide had synergistic effects with matrine enhancing matrine-induced apoptosis.

Successful use of rituximab in platelet transfusion refractoriness in a multi-transfused patient with myelodysplastic syndrome

Abstract A 61-year-old man with newly diagnosed INT-1 risk myelodysplastic syndrome – refractory cytopenia with multilineage dysplasia (MDS-RCMD) was not responsive to treatment, such as androgen, thalidomide, granulocyte – colony stimulating factor (G-CSF) combined with erythropoietin (EPO), interleukin-11 (IL-11) and thrombopoietin (TPO), and became transfusion dependent. Due to repeated blood transfusions, he developed platelet transfusion refractoriness (PTR) to platelets from cross-matched donors as well as random donors. Anti-HLA class I antibodies were positive with enzyme-linked immunosorbent assay; however, HLA-compatible platelet products were unavailable. PTR was unresponsive to high-dose immunoglobulin and plasma exchange. The patient was then treated with rituximab 375 mg/m2 on days 1 and 8, and 100 mg total dose on days 15 and 22. Already after the first dose of rituximab, the patient was able to received successful platelet transfusion from all donors. Therefore rituximab may be considered as a potential therapy for PTR.

Matrine cooperates with all-trans retinoic acid on differentiation induction of all-trans retinoic acid-resistant acute promyelocytic leukemia cells (NB4-LR1): possible mechanisms.

Retinoic acid resistance results in refractory disease, and recovery in acute promyelocytic leukemia remains a challenge in clinical practice, with no ideal chemotherapeutic drug currently available. Here we report on the effect of an active compound of Sophora flavescens called matrine (0.1 mmol/L) combined with all-trans retinoic acid (1 µmol/L) in alleviating retinoic acid resistance in acute promyelocytic leukemia-derived NB4-LR1 cells by differentiation induction, as can be seen by an induced morphology change, increased CD11b expression, and nitro blue tetrazolium reduction activity, and a decreased expression of the promyelocytic leukemia-retinoic acid receptor α fusion gene and protein product. We further explored the probable mechanism of how matrine promotes the recovery of differentiation ability in NB4-LR1 cells when exposed to all-trans retinoic acid. We observed that the combination of all-trans retinoic acid and matrine can increase the level of cyclic adenosine monophosphate and protein kinase A activity, reduce telomerase activity, and downregulate the protein expression of topoisomerase II beta in NB4-LR1 cells. The results of this study suggest the possible clinical utility of matrine in the treatment of retinoic acid-resistant acute promyelocytic leukemia.

Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.

Effect of Chinese medicine treatment based on pattern identification on cellular immunophenotype of myelodysplastic syndrome

ObjectiveTo observe the influence of treatment based on Chinese medicine pattern identification on cellular immunophenotype of the myelodysplastic syndrome (MDS).MethodsSixty patients with MDS were randomly and equally assigned to the treatment group and the control group using a randomized digital table. Thirty patients in each group included 3 risk levels (low, moderate and high risks) with each level 10 patients according to the international prognostic scoring system. The control group was given conventional therapy which was also used in the treatment group. While the treatment group was given Zuogui Pill (左归丸) and Yougui Pill (右归丸) for low risk patients; Qingwen Baidu Decoction (清瘟败毒饮) and Bazhen Decoction (八珍汤) for moderate risk patients; Gexia Zhuyu Decoction (膈下逐瘀汤) and Qinghao Biejia Decoction (青蒿鳖甲汤) combined with Shiquan Dabu Decoction (十全大补汤) for high risk patients. After the treatment, the differences of overall response rate and immunophenotype (CD13, CD14, CD15, CD33 and CD34) of each group were analyzed.ResultsThe overall response rate of the treatment group was significantly higher than the control group in low risk and moderate risk patients (P=0.029), there was no statistical differences of overall response rate between the treatment group and the control group in high risk patients (P=0.089). The expressions of CD13, CD14, CD33 and CD34 in all three risk levels of the treatment group were obviously decreased after the treatment, while CD15 in all three risk levels of the treatment group was obviously increased after the treatment (P<0.05 or P<0.01). Meanwhile, the difference values of CD13 and CD33 in low risk level of the treatment group, CD33 and CD34 in moderate risk level of the treatment group as well as CD34 and CD15 in high risk level of the treatment group, were all greater than the control groups and they were statistically significant (P<0.05 or P<0.01).ConclusionsIt shows a better therapeutic effect if the MDS patients treated with Chinese medicine pattern identification in addition to conventional therapy. Since the treatment may inhibit the malignant clones and improve the dysmaturity of granulocyte differentiation, it is a feasible option in clinical practice.