Thymoquinone Enhances Apoptosis of K562 Chronic Myeloid Leukemia Cells through Hypomethylation of SHP-1 and Inhibition of JAK/STAT Signaling Pathway.

The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML). SHP-1 functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement of SHP-1 expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent of Nigella sativa seeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression of SHP-1 through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status of SHP-1 was studied by pyrosequencing analysis. The expression of SHP-1, TET2, WT1, DNMT1, DNMT3A, and DNMT3B was determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated the DNMT1 gene, DNMT3A gene, and DNMT3B gene and upregulated the WT1 gene and TET2 gene. This led to hypomethylation and restoration of SHP-1 expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.

Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells.

Constitutive activation of Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways plays a crucial role in the development of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Thymoquinone (TQ), one of the main constituents of Nigella sativa, has shown anti-cancer activities in several cancers. However, the inhibitory effect mechanism of TQ on leukemia has not been fully understood. Therefore, this study aimed to investigate the effect of TQ on JAK/STAT and PI3K/Akt/mTOR pathways in MV4-11 AML cells and K562 CML cells. FLT3-ITD positive MV4-11 cells and BCR-ABL positive K562 cells were treated with TQ. Cytotoxicity assay was assessed using WSTs-8 kit. The expression of the target genes was evaluated using RT-qPCR. The phosphorylation status and the levels of proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways were investigated using Jess western analysis. TQ induced a dose and time dependent inhibition of K562 cells proliferation. TQ significantly downregulated PI3K, Akt, and mTOR and upregulated PTEN expression with a significant inhibition of JAK/STAT and PI3K/Akt/mTOR signaling. In conclusion, TQ reduces the expression of PI3K, Akt, and mTOR genes and enhances the expression of PTEN gene at the mRNA and protein levels. TQ also inhibits JAK/STAT and PI3K/Akt/mTOR pathways, and consequently inhibits proliferation of myeloid leukemia cells, suggesting that TQ has potential anti-leukemic effects on both AML and CML cells.

Gene Expression Profiling and Protein Analysis Reveal Suppression of the C-Myc Oncogene and Inhibition JAK/STAT and PI3K/AKT/mTOR Signaling by Thymoquinone in Acute Myeloid Leukemia Cells.

Overexpression of c-Myc plays an essential role in leukemogenesis and drug resistance, making c-Myc an attractive target for cancer therapy. However, targeting c-Myc directly is impossible, and c-Myc upstream regulator pathways could be targeted instead. This study investigated the effects of thymoquinone (TQ), a bioactive constituent in Nigella sativa, on the activation of upstream regulators of c-Myc: the JAK/STAT and PI3K/AKT/mTOR pathways in HL60 leukemia cells. Next-generation sequencing (NGS) was performed for gene expression profiling after TQ treatment. The expression of c-Myc and genes involved in JAK/STAT and PI3K/AKT/mTOR were validated by quantitative reverse transcription PCR (RT-qPCR). In addition, Jess assay analysis was performed to determine TQ's effects on JAK/STAT and PI3K/AKT signaling and c-Myc protein expression. The results showed 114 significant differentially expressed genes after TQ treatment (p < 0.002). DAVID analysis revealed that most of these genes' effect was on apoptosis and proliferation. There was downregulation of c-Myc, PI3K, AKT, mTOR, JAK2, STAT3, STAT5a, and STAT5b. Protein analysis showed that TQ also inhibited JAK/STAT and PI3K/AKT signaling, resulting in inhibition of c-Myc protein expression. In conclusion, the findings suggest that TQ potentially inhibits proliferation and induces apoptosis in HL60 leukemia cells by downregulation of c-Myc expression through inhibition of the JAK/STAT and PI3K/AKT signaling pathways.

Thymoquinone Inhibits Growth of Acute Myeloid Leukemia Cells through Reversal SHP-1 and SOCS-3 Hypermethylation: In Vitro and In Silico Evaluation.

Epigenetic silencing of tumor suppressor genes (TSGs) plays an essential role in cancer pathogenesis, including acute myeloid leukemia (AML). All of SHP-1, SOCS-1, and SOCS-3 are TSGs that negatively regulate JAK/STAT signaling. Enhanced re-expression of TSGs through de-methylation represents a therapeutic target in several cancers. Thymoquinone (TQ) is a major component of Nigella sativa seeds with anticancer effects against several cancers. However, the effects of TQ on DNA methylation are not entirely understood. This study aimed to evaluate the ability of TQ to re-express SHP-1, SOCS-1, and SOCS-3 in MV4-11 AML cells through de-methylation. Cytotoxicity, apoptosis, and cell cycle assays were performed using WSTs-8 kit, Annexin V-FITC/PI apoptosis detection kit, and fluorometric-red cell cycle assay kit, respectively. The methylation of SHP-1, SOCS-1, and SOCS-3 was evaluated by pyrosequencing analysis. The expression of SHP-1, SOCS-1, SOCS-3, JAK2, STAT3, STAT5A, STAT5B, FLT3-ITD, DNMT1, DNMT3A, DNMT3B, TET2, and WT1 was assessed by RT-qPCR. The molecular docking of TQ to JAK2, STAT3, and STAT5 was evaluated. The results revealed that TQ significantly inhibited the growth of MV4-11 cells and induced apoptosis in a dose- and time-dependent manner. Interestingly, the results showed that TQ binds the active pocket of JAK2, STAT3, and STAT5 to inhibit their enzymatic activity and significantly enhances the re-expression of SHP-1 and SOCS-3 through de-methylation. In conclusion, TQ curbs MV4-11 cells by inhibiting the enzymatic activity of JAK/STAT signaling through hypomethylation and re-expression of JAK/STAT negative regulators and could be a promising therapeutic candidate for AML patients.

Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells.

OBJECTIVE: BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells. METHODS: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis. RESULTS: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001). CONCLUSION: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Reduced hepcidin expression enhances iron overload in patients with HbE/ß-thalassemia: Α comparative cross-sectional study.

Iron homeostasis is regulated by hepcidin (HEPC) that controls the dietary iron absorption and iron recycling. HEPC deficiency contributes to iron overload in ß-thalassemia patients. The present study aimed to investigate the correlation between HEPC concentration and serum iron status among hemoglobin E (HbE)/ß-thalassemia patients and their parents (HbE trait and ß-thalassemia trait) compared with healthy controls. This study is a comparative cross-sectional study in which iron profile and HEPC level were examined in 65 HbE/ß-thalassemia patients (pretransfusion) and 65 parents at the Hospital Sultanah Nur Zahirah and in 130 students as healthy controls from Univesiti Sultan Zainal Abidin, Terengganu, Malaysia. Furthermore, six samples from each group (HbE/ß-thalassemia patients, parents and healthy controls) were randomly selected for gene expression analysis of HEPC and ferroportin1 (FPN1) using reverse transcription quantitative PCR. The results demonstrated that serum HEPC level were significantly decreased in HbE/ß-thalassemia patients and their parents (P<0.001) compared with healthy controls. In addition, the gene expression analysis showed a dramatically downregulated HEPC in HbE/ß-thalassemia patients and their parents (P=0.001) compared with healthy controls. However, there was a marked upregulation of FPN1 in HbE/ß-thalassemia patients and their parents (P=0.001) compared with healthy controls. Iron profiling results revealed a significantly increased serum ferritin in HbE/ß-thalassemia patients and their parents compared with healthy controls (P<0.001). In summary, the present study demonstrated that HEPC expression level and serum level were significantly decreased in HbE/ß-thalassemia patients and their parents, which was combined with a marked increased FPN1 expression level and serum ferritin level compared with healthy volunteers. These findings supported the hypothesis that downregulated HEPC could lose its function as a negative regulator of FPN1, resulting in iron overload in HbE/ß-thalassemia patients. Subsequently, assessing HEPC and FPN1 gene expression may be a useful tool to determine the risk of iron toxicity in patients with HbE/ß-thalassemia and their parents, and could therefore be considered as a therapeutic target in the management of iron burden in these patients.

Comparative proteomic analysis of different stages of breast cancer tissues using ultra high performance liquid chromatography tandem mass spectrometer.

BACKGROUND: Breast cancer is the fifth most prevalent cause of death among women worldwide. It is also one of the most common types of cancer among Malaysian women. This study aimed to characterize and differentiate the proteomics profiles of different stages of breast cancer and its matched adjacent normal tissues in Malaysian breast cancer patients. Also, this study aimed to construct a pertinent protein pathway involved in each stage of cancer. METHODS: In total, 80 samples of tumor and matched adjacent normal tissues were collected from breast cancer patients at Seberang Jaya Hospital (SJH) and Kepala Batas Hospital (KBH), both in Penang, Malaysia. The protein expression profiles of breast cancer and normal tissues were mapped by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Gel-Eluted Liquid Fractionation Entrapment Electrophoresis (GELFREE) Technology System was used for the separation and fractionation of extracted proteins, which also were analyzed to maximize protein detection. The protein fractions were then analyzed by tandem mass spectrometry (LC-MS/MS) analysis using LC/MS LTQ-Orbitrap Fusion and Elite. This study identified the proteins contained within the tissue samples using de novo sequencing and database matching via PEAKS software. We performed two different pathway analyses, DAVID and STRING, in the sets of proteins from stage 2 and stage 3 breast cancer samples. The lists of molecules were generated by the REACTOME-FI plugin, part of the CYTOSCAPE tool, and linker nodes were added in order to generate a connected network. Then, pathway enrichment was obtained, and a graphical model was created to depict the participation of the input proteins as well as the linker nodes. RESULTS: This study identified 12 proteins that were detected in stage 2 tumor tissues, and 17 proteins that were detected in stage 3 tumor tissues, related to their normal counterparts. It also identified some proteins that were present in stage 2 but not stage 3 and vice versa. Based on these results, this study clarified unique proteins pathways involved in carcinogenesis within stage 2 and stage 3 breast cancers. CONCLUSIONS: This study provided some useful insights about the proteins associated with breast cancer carcinogenesis and could establish an important foundation for future cancer-related discoveries using differential proteomics profiling. Beyond protein identification, this study considered the interaction, function, network, signaling pathway, and protein pathway involved in each profile. These results suggest that knowledge of protein expression, especially in stage 2 and stage 3 breast cancer, can provide important clues that may enable the discovery of novel biomarkers in carcinogenesis.