Gaillardin exerts potent antileukemic effects on HL-60 cells and intensifies arsenic trioxide cytotoxicity: Providing new insight into sesquiterpene lactones in leukaemia treatment.

The use of all-trans retinoic acid and arsenic trioxide resulted in favourable therapeutic responses in standard-risk acute promyelocytic leukaemia (APL) patients. However, resistance to these agents has made treating the high-risk subgroup more problematic, and possible side effects limit their clinical dosages. Numerous studies have proven the cytotoxic properties of Gaillardin, one of the Inula oculus-christi-derived sesquiterpene lactones. Due to the adverse effects of arsenic trioxide on the high-risk subgroup of APL patients, we aimed to assess the cytotoxic effect of Gaillardin on HL-60 cells as a single or combined-form approach. The results of the trypan blue and MTT assays outlined the potent cytotoxic properties of Gaillardin. The flow cytometric analysis and the mRNA expression levels revealed that Gaillardin attenuated the proliferative capacity of HL-60 cells through cell cycle arrest and induced apoptosis via reactive oxygen species generation. Moreover, the results of synergistic experiments indicated that this sesquiterpene lactone sensitizes HL-60 cells to the cytotoxic effects of arsenic trioxide. Taken together, the findings of the present investigation highlighted the antileukemic characteristics of Gaillardin by inducing G1 cell cycle arrest and triggering apoptosis. Gaillardin acts as an antileukemic metabolite against HL-60 cells and this study provides new insight into treating APL patients, especially in the high-risk subgroup.

Britannin, a sesquiterpene lactone induces ROS-dependent apoptosis in NALM-6, REH, and JURKAT cell lines and produces a synergistic effect with vincristine.

BACKGROUND: Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attraction in the therapeutic fields due to its anti-tumor properties. This study was designed to evaluate the effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). METHODS AND RESULTS: To determine the anti-leukemic effects of Britannin on ALL-derived cell lines and suggest a mechanism of action for the agent, we used MTT assay, Annexin-V/PI staining, ROS assay, and real-time PCR analysis. Moreover, by using a combination index (CI), we evaluated the synergistic effect of Britannin on Vincristine. We found that unlike normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells, Britannin reduced the viability of NALM-6, REH, and JURKAT cells. Among tested cells, NALM-6 cells had the highest sensitivity to Britannin, and this agent was able to induce p21/p27-mediated G1 cell cycle arrest and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in this cell line. When NALM-6 cells were treated with Nacetyl-L-Cysteine (NAC), a scavenger of ROS, Britannin could induce neither apoptosis nor reduce the survival of the cells suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. CONCLUSIONS: Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.

Ergolide, a potent sesquiterpene lactone induces cell cycle arrest along with ROS-dependent apoptosis and potentiates vincristine cytotoxicity in ALL cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Inula oculus christi belongs to the family of Asteraceae and it was traditionally wide used in treatment of kidney stones and urethra infection; besides, recently the potent sesquiterpene lactones isolated from inula species has gained increasing attention in cancer treatments. This study investigates the anti-cancer properties and underlying mechanism of ergolide isolated from Inula oculus christi against leukemic cell lines. METHODS: Viability, metabolic activity and proliferation evaluated using different index of MTT assay such as IC50 and GI50. Human erythrocytes were used to evaluate hemolytic activity. Flow-cytometry was used to detect and measure ROS level, and the induction of apoptosis and autophagy were evaluated using Annexin V/PI, Acridine Orange staining, respectively. Moreover, qRT-PCR was performed to examine the expression of a large cohort of crucial regulatory genes. Tunel assay was also carried out to assess morphologically ergolide effects. RESULTS: Ergolide did not exert ant cytotoxicity against non-tumorous cells and did not cause noticeable hemolysis. It also caused ROS production during early hours after treatment of cells which was then followed by cell cycle arrest in G0/G1 phase and autophagy induction. Using N-acetyl-L-cysteine (NAC), we found that ergolide could not increase ROS and induce autophagy and moreover repressed cell death, indicating that ergolide induce cell death through ROS-dependent manner by altering the expression of pro apoptotic related genes. Autophagy inhibition also potentiated ergolide-induced cell death. Furthermore, ergolide intensified vincristine cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines revealed robust synergistic properties of ergolide with VCR. CONCLUSION: Here we showed that ergolide could be considered as a potent natural compound against leukemic cells by inducing cell cycle arrest followed by dose-dependent cell death. Based on results, Autophagy response in a result of ROS accumulation acted as a survival pathway and blocking this pathway could noticeably increase ergolide cytotoxicity on ALL cell lines.

Methanolic Extract from Aerial Parts of Artemisia Annua L. Induces Cytotoxicity and Enhances Vincristine-Induced Anticancer Effect in Pre-B Acute Lymphoblastic Leukemia Cells.

Background: Nowadays, remarkable attention has been drawn towards the effective therapeutic characteristic of natural products targeting cancerous cells. This study aimed to investigate the anti-cancer effect of Artemisia annua extract (AAE), a Chinese herbal medicine alone and in combination with a microtubule binding agent used in ALL treatment, vincristine (VCR), in B-Acute lymphoblastic leukemia (ALL) Nalm-6 and Reh cells. Materials and Methods: Cytotoxic activity of AAE and VCR was determined using MTT assay in Nalm-6, and Reh cell lines and synergism was evaluated using the CompuSyn software. Caspase 3 activity and Annexin/PI staining were performed for apoptosis assessment. The expression level of apoptosis-related genes, caspase 3, Bax and Bcl-2 were determined using real time-PCR. One-way ANOVA and post hoc Tukey multiple comparisons were used for statistical analysis. Results: Our findings revealed that a single administration of AAE exerted an anti-leukemic effect in both ALL-derived cells in a time- and dose-dependent manner. Interestingly, the growth inhibitory activity of the extract was more potentiated when combined with 0.1 and 1 nM VCR through caspase 3-dependent apoptosis. Moreover, real-time PCR analysis showed that VCR-induced cytotoxicity was augmented by AAE through alteration of Bax, and Bcl-2 mRNA expression. Conclusion: Overall, owing to the nontoxic nature of AAE and its explicit role in enhancing VCR effectiveness, our study provided new insight into the development of a novel combinatorial approach in ALL using natural herbs. The practical implication of the research requires further investigation through clinical trials, opening avenues for forthcoming treatment improvements.

The effect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have accelerated atherosclerosis as a pro thrombotic state that is associated with the platelet activation priming. Platelets, which undergo the continuous mild stimulation, may lose their sensitivity to react to a strong stimulation. The present study aimed to investigate activation responses of platelets to mild and subsequent strong stimulations in patients with T2DM and healthy individuals. METHODS: Blood samples, which were taken from 40 patients with T2DM and 35 healthy individuals, were collected into the citrate containing tubes. The samples were subjected to the soft centrifugation to prepare the platelet rich plasma (PRP). Platelets in PRP samples were treated at a low (1 µM) concentration and then at a high (10 µM) concentration of ADP. Before and after stimulation with different doses of ADP, levels of CD62P expression and formation of platelet micro particles (PMPs) were measured using a flow cytometry method. RESULTS: The platelets from patients with T2DM had higher levels of CD62P expression before any stimulation (P = 0.003) than control samples. Platelets, which underwent the mild stimulation, indicated lower responses to CD62P expression, but higher PMPs formation after stimulation with high dose of ADP. Patients with T2DM had higher platelet micro particles in all states with the ADP stimulation. (P = 0.004, SD: ±74.52). CONCLUSIONS: The flow cytometry data indicated that platelets were pre-active and associated with metabolic conditions in patients with type 2 diabetes mellitus. The induction of desensitization state helped platelets to reduce the platelet activation and sensitivity to ADP in a diabetic environment. Furthermore, the production of platelets micro-particles was high in the patients; and desensitized platelets were more susceptible to shedding of micro-particles.

Centaurea albonitens extract enhances the therapeutic effects of Vincristine in leukemic cells by inducing apoptosis.

Drug-induced toxicities and dose-related side effects are the major challenges in the conventional cancer therapy by the chemo drugs. On the other hand, herbal derivatives have obtained a great research interest in the field of therapeutic applications because of their more favorable specifications including less toxicity, cost-effective and more physiologically compatible than the chemical drugs. For this purpose, we evaluated methanolic extract prepared from Centaurea albonitens Turrill alone and in combination with Vincristine (VCR) for its potential cytotoxic effects in NALM-6, REH, NB4 and KMM-1 cell lines by using the various approaches. Centaurea genus is one of the current medicinal plants, which has used in traditional medicine, However, there are rare studies to examine its anticancer properties against hematologic malignant cells. In this study, we demonstrated Centaurea albonitens extract (CAE) induces cytotoxicity through G0/G1 phase arrest followed by apoptosis in a dose- and time- dependent manner, although with varying efficiency. Interestingly, normal cells didn't exhibit significant cytotoxicity after CAE treatment. Moreover, we found that low dose of CAE enhances anti-cancer effects of VCR in pre-B ALL cell lines (NALM-6 and REH). Further investigations validated synergistic anticancer activities of VCR and CAE through inducing apoptosis without significant cell cycle arrest. Taken together, our results demonstrated for the first time that the methanolic extract of Centaurea albonitens can be considered as a potential anticancer agent and/or an enhancer of chemotherapeutic sensitivity of VCR.

Effect of autologous salvaged blood on postoperative natural killer cell precursor frequency.

BACKGROUND: Immunosuppression after major surgery increases the risk of infections. Natural killer cells play a pivotal part in defence against infection. We aimed to investigate the immunomodulatory effects of different types of postoperative blood transfusion by use of a new assay for measuring the frequency of peripheral blood natural killer precursor cells (NKpf assay). METHODS: We measured the natural killer cell precursor (NKp) frequency before and 5 days after surgery in 120 patients undergoing joint replacement surgery. The patients were assigned to one of five groups according to the type of transfusion received: non-transfused (n=32), allogeneic non-leukodepleted blood (eight), allogeneic leukodepleted blood (30), autologous predeposited blood (ten), and autologous salvaged blood collected within the first 24 h after surgery (40). We also measured interferon gamma and interleukin 10 concentrations before and after surgery. FINDINGS: The mean postoperative NKp frequency for all patients was lower than the preoperative values, except in patients receiving autologous salvaged blood, which was higher than all other groups (p<0.0001). Postoperative NKp frequencies for patients receiving allogeneic or autologous predeposited blood responded similarly (p=0.99), but these patients had lower NKp frequencies than did the non-transfused group (p<0.0001). Postoperative interferon gamma concentrations were higher in the autologous salvaged blood group (p<0.0001) than in other groups, which did not differ from each other. Interleukin 10 concentrations were similar across all groups (p=0.49). INTERPRETATION: Immunosuppression associated with surgery and blood loss was reflected in a reduced frequency of NKp and decreased interferon gamma. This immunosuppression was reversed by transfusion of autologous salvaged blood, suggesting that this fluid contained immunostimulants.