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Neosetophomone B (NSP–B), a meroterpenoid fungal secondary metabolite, was investigated for its anticancer potential in leukemic cell lines (K562 and U937). NSP-B treatment of leukemic cells suppressed cell viability by triggering apoptotic cell death. Apoptosis induced by NSP-B is triggered by mitochondrial signaling and caspase activation. Additionally, NSP-B treatment of leukemic cells causes AKT's inactivation accompanied by downregulation of SKP2 oncogene and MTH1 with a concomitant increase of p21Cip1and p27Kip1. Furthermore, NSP-B causes suppression of antiapoptotic proteins, including cIAP1, cIAP2, XIAP, survivin and BCl-XL. Overall, NSP-B reduces cell viability by mitochondrial and caspase-dependent apoptosis. The inhibition of AKT and SKP2 axis could be a promising therapeutic target for leukemia treatment. This work was supported by grant funded by the Medical Research Center (MRC), Hamad Medical Corporation, Doha, Qatar (MRC-01-21-301). The authors thank Qatar National Library for open access support of this article.

Multiple myeloma (MM) is a hematological disorder characterized by the abnormal expansion of plasma cells in the bone marrow. Despite great advances over the past three decades in discovering the efficacious therapies for MM, the disease remains incurable for most patients owing to emergence of drug-resistant cancerous cells. Guggulsterone (GS), a phytosteroid, extracted from the gum resin of guggul plant, has displayed various anticancer activities in vitro and in vivo; however, the molecular mechanisms of its anticancer activity have not been evaluated in MM cells. Therefore, in this study, we investigated the anticancer activity of GS in various MM cell lines (U266, MM.1S, and RPMI 8226) and the mechanisms involved. GS treatment of MM cells caused inhibition of cell proliferation and induction of apoptotic cell death as indicated by increased Bax protein expression, activation of caspases, and cleavage of poly (ADP-ribose) polymerase. This was associated with the downregulation of various proliferative and antiapoptotic gene products, including cyclin D, Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. GS also suppressed the constitutive and interleukin 6-induced activation of STAT3. Interestingly, the inhibition of Janus activated kinase or STAT3 activity by the specific inhibitors or by siRNA knockdown of STAT3 resulted in the downregulation of HMGB1, suggesting an association between GS, STAT3, and HMGB1. Finally, GS potentiated the anticancer effects of bortezomib (BTZ) in MM cells. Herein, we demonstrated that GS could be a potential therapeutic agent for the treatment of MM, possibly alone or in combination with BTZ. Medical Research Center, Grant no MRC-01-18-120 (SU) and MRC-01-20-858 (KSP), Hamad Medical Corporation, Doha, Qatar. The Open Access funding is provided by Qatar National Library.

Effective treatment of lung cancer remains a significant clinical challenge due to its multidrug resistance and side effects of the current treatment options. The high mortality associated with this malignancy indicates the need for new therapeutic interventions with fewer side effects. Natural compounds offer various benefits such as easy access, minimal side effects, and multi-molecular targets and thus, can prove useful in treating lung cancer. Sanguinarine (SNG), a natural compound, possesses favorable therapeutic potential against a variety of cancers. Here, we examined the underlying molecular mechanisms of SNG in Non-Small Cell Lung Cancer (NSCLC) cells. SNG suppressed cell growth and induced apoptosis via downregulation of the constitutively active JAK/STAT pathway in all the NSCLC cell lines. siRNA silencing of STAT3 in NSCLC cells further confirmed the involvement of the JAK/STAT signaling cascade. SNG treatment increased Bax/Bcl-2 ratio, which contributed to a leaky mitochondrial membrane leading to cytochrome c release accompanied by caspase activation. In addition, we established the antitumor effects of SNG through reactive oxygen species (ROS) production, as inhibiting ROS production prevented the apoptosis-inducing potential of SNG. In vivo xenograft tumor model further validated our in vitro findings. Overall, our study investigated the molecular mechanisms by which SNG induces apoptosis in NSCLC, providing avenues for developing novel natural compound-based cancer therapies.

Cancer is one of the most life threatening diseases, causing nearly 13% death in the worldwide. Leukemia, cancer of the hematopoetic cells is the main cause of cancer death in adults and children. Therapeutic agents used in treatment of cancer are known to have narrow therapeutic window and tendency to develop resistance against some cancer cell lines thus, proposing a need to discover some novel agents to treat cancer. In the present study we investigated the anticancer activity of Neosetophomone B(NSP-B), an aquatic fungal metabolite isolated from Neosetophoma sp against leukemic cells (K562 and U937). MTT results demonstrated a dose dependent inhibition of cell proliferation in K562 and U937 cell lines. Annexin staining using flow cytometry indicated that NSP-B treatment cause a dose dependent apoptosis in leukemic cells.Western blot analysis showed that NSP-B mediated apoptosis involves sequential activation of caspase 9, 3 and poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore NSP-B treatment of leukemic cells resulted in upregulation of pro-apoptotic proteins (Bax) with downregulation of anti-apoptotic proteins ( Bcl-2 ).Thus, present study focuses on exploring the mechanism of anticancer activity of NSP-B on leukemic cells, raising the possibility of its use as a novel therapeutic agent for hematological malignancies. Results: We sought to determine whether NSP-B suppresses the growth of leukemic cell lines. We tested a panel of leukemic cell lines with different doses of NSP-B. Cell viability decreased in a concentration-dependent manner in K562 and U937 cell lines. NSP-B induced apoptosis in K562 and U937 cell lines via downregulation of anti-apoptotic proteins and enhancement of pro-apoptotic proteins. NSP-B induced the activation of caspase cascade signaling pathway. Altogether our results suggest that the NSP-B plays an important role in apoptosis in leukemic cell lines .Conclusions: Our data provides insight on anticancer activities of NSP-B in leukemic cell lines (K562 and U937). NSP-B inhibit cell viability via inducing apoptosis. The NSP-B mediated apoptosis occurs via downregulation of anti-apoptotic proteins and enhancement of pro-apototic proteins, thereby activating the caspase-cascade signaling. Further studies are required to elicit role of NSP-B in regulating molecular pathway involved in the progression of cancer. Taken together, above results suggest that NSP-B may have a future therapeutic role in leukemia and possibly other hematological malignancies.

Forkhead box M1 (FoxM1) is a transcription factor that plays an important role in the etiology of many cancers, however, its role has not been elucidated in B-precursor acute lymphoblastic leukemia (B-pre-ALL). In the current study, we showed that the downregulation of FoxM1 by its inhibitor thiostrepton inhibited cell viability and induced caspase-dependent apoptosis in a panel of B-pre-ALL cell lines. Thiostrepton led downregulation of FoxM1 accompanied by decreased expression of Aurora kinase A, B, matrix metalloproteinases, and oncogene SKP2 as well as MTH1. Downregulation of the FoxM1/SKP2/MTH1 axis led to increase in the Bax/Bcl2 ratio and suppression of antiapoptotic proteins. Thiostrepton-mediated apoptosis was prevented by N-acetyl cysteine, a scavenger of reactive oxygen species. Co-treatment of B-pre-ALL with subtoxic doses of thiostrepton and bortezomib potentiated the proapoptotic action. Altogether, our results suggest that targeting FoxM1expression could be an attractive strategy for the treatment of B-pre-ALL. This work was supported by the grants funded by Medical Research center (MRC), Hamad Medical corporation, Doha, Qatar (MRC# 16354/16).

Sanguinarine (SNG), a natural compound with an array of pharmacological activities, has promising therapeutic potential against a number of pathological conditions, including malignancies. In the present study, we have investigated the antiproliferative potential of SNG against two well-characterized papillary thyroid cancer (PTC) cell lines, BCPAP and TPC-1. SNG significantly inhibited cell proliferation of PTC cells in a dose and time-dependent manner. Western blot analysis revealed that SNG markedly attenuated deregulated expression of p-STAT3, without affecting total STAT3, and inhibited growth of PTC via activation of apoptotic and autophagy signaling cascade, as SNG treatment of PTC cells led to the activation of caspase-3 and caspase-8, cleavage of PARP and activation of autophagy markers. Further, SNG-mediated anticancer effects in PTC cells involved the generation of reactive oxygen species (ROS) as N-acetyl cysteine (NAC), an inhibitor of ROS, prevented SNG-mediated antiproliferative, apoptosis and autophagy inducing action. Interestingly, SNG also sensitized PTC cells to chemotherapeutic drug cisplatin, which was inhibited by NAC. Finally, SNG suppressed the growth of PTC thyrospheres and downregulated stemness markers ALDH2 and SOX2. Altogether, the findings of the current study suggest that SNG has anticancer potential against PTC cells as well its derived cancer stem-like cells, most likely via inactivation of STAT3 and its associated signaling molecules.

Natural products have been gaining recognition and are becoming a significant part of research in the area of drug development and discovery. Phytochemicals derived from these sources have been comprehensively studied and have displayed a wide range of activities against many fatal diseases including cancer. One such product that has gained recognition from its pharmacological properties and nontoxic nature is embelin, obtained from Embelia ribes. Amid all the vivid pharmacological activities, embelin has gained its prominence in the area of cancer research. Embelin binds to the BIR3 domain of XIAP, preventing the association of XIAP and caspase-9 resulting in the suppression of cell growth, proliferation and migration of various types of cancer cells. Furthermore, embelin modulates anti-apoptotic pathways by suppressing the activity of NF-κB, PI3-kinase/AKT, JAK/STAT pathway – among others. The present review summarizes the various reported effects of embelin on different types of cancer cells and highlights the cellular mechanisms of action.

Sanguinarine (Sang), a plant-derived compound isolated from the roots of Sanguinaria canadensis was evaluated for its potential pro-apoptotic effects in precursor B acute lymphoblastic leukemia (Pre-ALL) cell lines. Treatment of 697, REH, RS4;11, and SupB15 cell lines with Sang exhibited significant inhibition of cell viability via induction of apoptotic cell death. Sang-mediated apoptosis was found to be associated with the increased expression of proapoptotic bax with concomitant decrease of Bcl-2 expression leading to depolarization of mitochondria membrane resulting in loss of mitochondrial membrane potential (MMP). The reduced MMP caused the leakage in mitochondrial membrane and release of cytochrome c into the cytosol. The cytochrome c then mediates the activation of caspase-cascade and subsequently PARP cleavage. Furthermore, pretreatment with z-VAD-FMK, a pan-caspase inhibitor, abrogated Sang-induced inhibition of cell viability, induction of apoptosis. Sang treatment also reduced the phosphorylation of AKT and suppressed the expression of a number of anti-apoptotic genes such as cIAP1, cIAP2, and XIAP. Sang mediates its anti-cancer activity by generation of reactive oxygen species (ROS) due to depletion of glutathione level in leukemic cell lines. Pretreatment of these cells with N-acetyl cysteine (NAC) prevented Sang-induced depletion of glutathione level and mitochondrial-caspase-induced apoptosis. Finally, Sang treatment of Pre-ALL cell suppressed colony formation ability of these cells suggesting Sang has an anti-leukemic potential. Altogether, our data suggest that Sang is an efficient inducer of intrinsic apoptotic cell death via generation of ROS and exhibition of anti-leukemic effect in Pre-ALL cells raises the possibility to develop Sang as a therapeutic modality for the treatment and management of Pre-ALL. This study was supported by Medical Research Center [RP # 16354/16], Hamad Medical Corporation, Doha, and State of Qatar. Scopus

Therapeutic agents used in the treatment of cancer are known to develop resistance against cancer cells. Hence, there is a continuing need to investigate novel agents for the treatment and management of cancer. Antitumor activity of greensporone C (GC), a new resorcylic acid lactone isolated from an organic extract of a culture of a Halenospora sp. freshwater fungus, was subjected for screening against a panel of leukemic cell lines (K562, U937, and AR320). In all the three cell lines, cell proliferation was inhibited in dose-dependent fashion. GC further arrested the cells in SubG0 phase in dose-dependent manner. Annexin V/PI dual staining data confirmed apoptotic death of treated K562 and U937 leukemic cells. Treatment with GC suppressed constitutively phosphorylated AKT and downregulated expression of inhibitor of apoptotic proteins XIAP, cIAP-1, and cIAP-2. In summation to this, GC-treated leukemic cells upregulated protein expression of pro-apoptotic proteins, Bax with concomitant decrease in expression of anti-apoptotic proteins including Bcl-2 and Bcl-xL. Upregulation of Bax was associated with cytochrome c release which was confirmed from the collapse of mitochondrial membrane. Released cytochrome c further activated caspase cascade which in turn initiated apoptosis process. Anticancer activity of this isolated fungal compound GC was potentiated via stimulating production of reactive oxygen species (ROS) along with depletion of reduced glutathione (GSH) levels in K562 and U937 leukemic cells. Pretreatment of these cells with N-acetyl cysteine prevented GC-induced depletion of reduced GSH level and mitochondrial-caspase-induced apoptosis. Altogether, our data show that GC modulates the apoptotic response of human leukemic cells and raises the possibility of its use as a novel therapeutic strategy for hematological malignancies. ? 2018 Prabhu, Siveen, Kuttikrishnan, Iskandarani, Khan, Merhi, Omri, Dermime, El-Elimat, Oberlies, Alali and Uddin. The authors would like to thank Queenie Fernandes and Sara Taleb for their technical support This work was funded and supported by Medical Research Centre (grant number: RP#16102/16), Hamad Medical Corporation, Doha, Qatar. Scopus

Background:Colorectal cancer (CRC) is the third most common cancer in Qatar and a major health concern for the Qatari population. Qatar has the highest rate of colon cancer compared to other countries in the eastern Mediterranean, West Asia and North Africa. Colon cancer is the most common cancer among Qatar's male population and according to the world age standard rates, around 20.8% of male Qataris have this cancer. Although progress in diagnosis and treatment has helped to extend and save the lives of many colorectal cancer patients, it still remains as one of the most prevalent human cancers. Many drugs such as 5-fluorouracil (5-FU) are being used to treat colorectal cancer, but patient(s) response to these drugs vary widely in terms of efficacy and toxicity. Moreover, it is observed that in colon cancer patients the tumor starts to develop resistance against these drugs over the course of treatment. The harmful side effects exhibited by the drugs used in cancer therapy as well as the increasing frequency of resistance to drugs have become the most challenging issues in the treatment of colorectal cancer. Hence, there exists an immediate need to discover better targeted and reliable drugs that could act as therapeutic agents which can prevent colon cancer progression and control distant metastasis as well as cure the disease with minimal side effects. Chemotherapeutic agents obtained from natural sources (plants) holds promising potential and have gained significant recognition in the field of cancer therapy. Pristimerin is a triterpenoid quinine methide present in various plant species of Cleastraceae and Hippocrateaceae families. Pristimerin has been shown to inhibit the proliferation of glioma, leukemia, myeloma, breast, lung, prostate and pancreatic cancer cell lines. Recent studies shows that pristimerin is a potent inhibitor of NF-κB. Induction of apoptosis by pristimerin was found to involve activation of caspases, mitochondrial dysfunction and inhibition of Akt signaling pathways. Pristimerin was reported to induce apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl.Material and MethodsReagentsAntibodies against Caspase 3, caspase-9, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) antibody was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit with BD GolgiPlug, Propidium Iodide Staining Solution, Annexin V Binding Buffer, Mitochondrial Membrane Potential Detection (JC-1) Kit, Stain Buffer (FBS), Annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, Rabbit Anti- Active Caspase-3- BV605 antibody and PARP Cleaved Form-AF700 antibody were obtained from BD Biosciences (NJ, USA). CellROX Deep Red Reagent, MitoSOX Red Mitochondrial Superoxide Indicator, SYTOX Blue Nucleic Acid Stain and Hoechst 33342 solution were obtained from Molecular Probes, Life Technologies (CA, USA). Pristimerin, Cell Counting Kit-8 (CCK-8), DAPI, Glutathione-Reduced (GSH) and N-Acetyl-L-Cysteine (NAC) were obtained from Sigma-Aldrich (MO, USA). Human Apoptosis Antibody Array and Phospho-Kinase Antibody Array were purchased from R&D systems (MN, USA). Cell Death Detection ELISAPLUS kit was purchased from Roche Diagnostics (Mannheim, Germany).MethodsCell culture: Human colorectal cancer cell line HCT116 was cultured in DMEM, OXCO1 and SW48 cells were cultured in RPMI 1640 medium. Both culture medium were supplemented with 10% Heat Inactivated fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin. Cells were cultured at 37°C under a humidified 95%:5% (v/v) mixture of air and CO2.Pristimerin was dissolved in dimethylsulfoxide as a 10 mM stock solution and stored at 4°C for the in vitro experiments. Further dilution was done in cell culture medium as required.Cell Viability: Effect on the viability of CRC cell lines, HCT116, OXCO1 and SW48 were determined following treatment with various doses of Pristimerin for 24, 48 and 72 hours using WST-8 kit.Apoptosis: HCT116/OXCO1 cells were treated with various doses of Pristimerin for 24 hours. After incubation, cells were harvested, washed with PBS and stained with Annexin V-FITC/PI for 20 minutes at room temperature and apoptosis was measured by flow cytometry using BD LSRFortessa analyzer (BD Biosciences, USA).H2AX, active caspase-3 and cleaved PARP were quantified by flow cytometry. After treatment with Pristimerin, HCT116 cells were fixed and permeabilized using BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, as per protocol from the manufacturer. 0.5 × 106 cells in Stain Buffer (FBS) were stained with 3 μL each of H2AX (pS139)-Alexa Fluor 647, Rabbit Anti- Active Caspase-3- BV605 and PARP Cleaved Form-AF700 antibodies for 30 minutes at room temperature. The cells were washed with Stain Buffer (FBS) and then analyzed by flow cytometry.Cell cycle analysis: 0.5 × 106 cells (HCT116/OXCO1) were briefly stained with Hoechst 33342 solution (10 μg/mL) and then analyzed by flow cytometry.Mitochondrial membrane potential: 0.5 × 106 cells (HCT116/OXCO1) were briefly stained with JC-1 stain for 15 minutes at 37°C as per instructions from the kit manufacturer. The cells were washed twice with 1x assay buffer and then analyzed by flow cytometry.ROS Production: CellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The signals from CellROX Deep Red Reagent is localized in the cytoplasm. The production of superoxide by mitochondria was quantitated using the MitoSOX Red reagent. It is rapidly oxidized by superoxide but not by other reactive oxygen species and reactive nitrogen species. HCT116/OXCO1 cells were treated with Pristimerin (0, 1, 2.5, 5 μM) for 24 h and finally analyzed by flow cytometry for quantification of ROS and superoxide.HCT116 cells were preincubated with NAC (2.5 mM) or GSH (5 mM) for 30 minutes before addition of Pristimerin (5 μM) in studies to confirm the role of ROS in induction of apoptosis.Western blot: Following treatment with various doses/combinations of Pristimerin for 24 hours, HCT116 cells were lysed with RIPA buffer and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies. Target proteins were detected using an enhanced chemiluminescence (Bio-Rad ChemiDoc MP imaging system).Statistics: Two-tailed Student's t tests was performed using the Origin Pro software to compare means of different treatment groups and a P value below 0.05 was considered statistically significant.ResultsThe results show that Pristimerin causes a dose dependent inhibition of cell proliferation in various CRC cell lines, HCT116, OXCO1 and SW48. The inhibition of proliferation correlated with the induction of apoptosis in HCT116 and OXCO1 cell lines. Treatment with Pristimerin leads to activation of Bid, loss of mitochondrial membrane potential, as determined by JC1 staining, activation of caspase-9, subsequent activation of caspase-3 followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage and DNA double strand breaks (H2AX staining). Pristimerin was found to increase the oxidative stress in CRC cells in a dose dependent manner. Pretreatment of HCT116 cells with NAC/GSH, was found to inhibit Pristimerin mediated induction of apoptosis, confirming the role of ROS.ConclusionAltogether, these data confirm the role of ROS in the inhibition of cell proliferation and the induction of apoptosis in CRC cell lines by Pristimerin, and thus provide a strong rationale for pursuing the detailed mechanism of action and confirming the anti cancer activity using in vivo models and future clinical studies.KeywordsColorectal cancer, Pristimerin, oxidative stress, apoptosis.

BackgroundThe X-linked inhibitor of apoptosis (XIAP) is a promising molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Embelin, identified primarily from the Embelica ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity.Material and MethodsReagentsEmbelin was purchased from Tocris (Cambridge, MA). TRAIL was purchased from Alexis Corporation (Lausen, Switzerland). Antibodies against Caspase 3, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, Propidium Iodide Staining Solution, Annexin V Binding Buffer, Mitochondrial Membrane Potential Detection (JC-1) Kit, Stain Buffer (FBS), Annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, Rabbit Anti- Active Caspase-3- BV605 antibody and PARP Cleaved Form-AF700 antibody were obtained from BD Biosciences (NJ, USA). CellROX Deep Red Reagent was obtained from Molecular Probes, Life Technologies (CA, USA). (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) solution (MTT) powder, DAPI, and N-Acetyl-L-Cysteine were obtained from Sigma-Aldrich (MO, USA). Apoptotic DNA ladder Kit was procured from Thermo fisher Scientific (USA).MethodologyWe used following assays and methods for this study.Cell cultureLeukemic cell lines K562 and U937 leukemic cells were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin at 370C in humidified atmosphere containing 5% CO2. All experiments were conducted in 5% serum.Cell viabilityExperiments were performed following treatment with various doses of embelin with and without pre-treatment with NAC for 24 hours using MTT assay.ROS ProductionCellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The cell-permeable reagent is non-fluorescent or very weakly fluorescent while in a reduced state and upon oxidation exhibit strong fluorogenic signal. The signals from CellROX Deep Red Reagent are localized in the cytoplasm and measured by flow cytometry (Excitation 640 nm/Emission 665 nm). K562 cells were treated with embelin for indicated time periods and finally analyzed by flow cytometry.ApoptosisApoptosis was measured using annexinV-FITC/PI staining and analyzed by flow cytometry. Cells were treated with embelin in the presence and absence of NAC for 24 hours. Following treatment, cells were harvested, washed with PBS and stained with annexin V-FITC/PI for 20 minutes at room temperature and apoptosis was measured by flow cytometry.Western blotFollowing treatment with embelin and NAC for 24 hours, cells were lysed and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies.ResultsThe results from our study showed that Embelin causes a dose dependent inhibition of cell proliferation in K562 and U937 leukemic cells. Anti-proliferative activity of Embelin correlated with induction of apoptosis. In addition, Embelin treatment of K562 cells decreased the constitutive phosphorylation/activation of AKT followed by the upregulation of proapototic protein Bax. Embelin also induced loss of mitochondrial membrane potential, as determined by JC1 staining, with subsequent activation of caspase-3 and polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. Pretreatment of K562 cells with N-acetyl-L-cystein, a scavenger of reactive oxygen species (ROS) prevented Embelin mediated apoptotic effects. Embelin also suppressed K562 derived progenitor colony formation, suggesting its antileukemic effect. Finally our data also showed that co-treatment of subtoxic doses of Embelin and TRAIL potentiated anticancer activity in leukemic cells.ConclusionAltogether, these findings suggest that Embelin causes inhibition of cell proliferation and induction of apoptosis via generation of ROS in leukemic cells, which raises the possibility that Embelin alone or in combination of chemotherapeutic agents may have a future therapeutic role in leukemia and possibly other malignancies with up-regulated XIAP pathway.KeywordsApoptosis, CML, ROS

The X-linked inhibitor of apoptosis (XIAP) is a viable molecular target for anticancer drugs that overcome apoptosis-resistance of malignant cells. XIAP is an inhibitor of apoptosis, mediating through its association with BIR3 domain of caspase 9. Embelin, a quinone derivative isolated from the Embelia ribes plant, has been shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity. In this study, we found that embelin causes a dose-dependent suppression of proliferation in leukemic cell lines K562 and U937. Embelin mediated inhibition of proliferation correlates with induction of apoptosis. Furthermore, embelin treatment causes loss of mitochondrial membrane potential and release of cytochrome c, resulting in subsequent activation of caspase-3 followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. In addition, embelin treatment of leukemic cells results in a decrease of constitutive phosphorylations/activation level of AKT and downregulation of XIAP. Gene silencing of XIAP and AKT expression showed a link between XIAP expression and activated AKT in leukemic cells. Interestingly, targeting of XIAP and PI3-kinase/AKT signaling augmented inhibition of proliferation and induction of apoptosis in leukemic cells. Altogether these findings raise the possibility that embelin alone or in combination with inhibitors of PI3-kinase/AKT pathway may have therapeutic usage in leukemia and possibly other malignancies with up-regulated XIAP pathway.

Background Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade®, a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML. Methods Cell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose. Results Treatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells. Conclusions Altogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0823-y) contains supplementary material, which is available to authorized users.