CDH1 overexpression sensitizes TRAIL resistant breast cancer cells towards rhTRAIL induced apoptosis.

PURPOSE: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is well known for its unique ability to induce apoptosis in cancer cells but not normal cells. However, a subpopulation of cancer cells exist that does not respond to toxic doses of TRAIL. In this study, we aimed to identify key factors regulating TRAIL resistance in breast cancer. METHODS: rhTRAIL (recombinant human TRAIL) resistant cells (TR) isolated from TRAIL sensitive MDA-MB-231 parental cells (TS) were confirmed using trypan blue assay, cell viability assay and AO/EtBr (acridine orange/ethidium bromide) staining. Microarray was performed followed by analysis using DAVID and Cytoscape bioinformatics software to identify the candidate hub gene. Gene expression of the candidate gene was confirmed using real-time PCR and western blot. Candidate gene was overexpressed via transient transfection to identify its significance in the context of rhTRAIL. Breast cancer patient data was obtained from The Cancer Genome Atlas (TCGA) database. RESULTS: Whole transcriptome analysis identified 4907 differentially expressed genes (DEGs) between TS and TR cells. CDH1 was identified as the candidate hub gene, with 18-degree centrality. We further observed CDH1 protein to be downregulated, overexpression of which increased apoptosis in TR cells after rhTRAIL treatment. TCGA patient data analysis also showed CDH1 mRNA to be low in TRAIL resistant patient group compared to TRAIL sensitive group. CONCLUSION: CDH1 overexpression sensitizes TR cells towards rhTRAIL induced apoptosis. Therefore, we can hypothesize that CDH1 expression should be taken into account while performing TRAIL therapy in breast cancer.

Identification of two novel thiophene analogues as inducers of autophagy mediated cell death in breast cancer cells.

Natural compounds isolated from different medicinal plants remain one of the major resources of anticancer drugs due to their enormous chemical diversity. Studies suggested therapeutic potential for various tanshinones, key bioactive lipophilic compounds from the root extracts of Salvia miltiorrhiza Bunge, against multiple cancers including breast carcinoma. We designed, synthesized and evaluated anti-cancer properties of a series of condensed and doubly condensed furophenanthraquinones of tanshinone derivatives on two breast cancer lines - MCF7 and MDA-MB-231. We identified two thiophene analogues - compounds 48 and 52 with greater anti-proliferative efficiency (~4 fold) as compared to the natural tanshinones. Mechanistically, we showed that both compounds induced autophagy mediated cell death and partial but significant restoration of cell death in the presence of autophagy inhibitor further supported this notion. Both compounds transcriptionally activated several autophagy genes responsible for autophagosome formation along with two death regulators - GADD34 and CHOP for inducing cell death. Altogether, our studies provide strong evidence to support compounds 48 and 52 as promising leads for further development as anticancer agents through modulating autophagy mechanism.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of cytokine superfamily, induces apoptosis in a number of tumor cells through the activation of extrinsic apoptotic pathway but shows little or no cytotoxicity toward normal cells. However some tumor cells are inherently resistant to TRAIL-mediated apoptosis, which needs to be addressed to establish TRAIL as a potential chemotherapeutic drug. In this study, our aim was to manipulate TRAIL-apoptosis pathway by hydroxychavicol (HCH), a polyphenol from Piper betel leaf, for the induction of apoptosis in TRAIL resistant chronic myeloid leukemia cell. When imatinib-resistant K562 cells were treated with HCH, it made these K562 cells sensitive to TRAIL. It was observed that HCH downregulated antiapoptotic proteins XIAP and FLIP, whereas the expression of TRAIL receptors, DR4 and DR5, remains unchanged. Moreover, we observed that reactive oxygen species or ROS played a crucial role in the downregulation of FLIP and XIAP because ROS scavenger significantly reversed the decrease of XIAP, and FLIP. Ubiquitin-proteasome pathway was observed to play a crucial role in the downregulation of XIAP and FLIP, as proteasomal inhibitor MG132 significantly reversed the downregulation of XIAP and FLIP. In conclusion, this study demonstrates the combinatorial treatment of TRAIL and HCH as promising alternative therapeutic approach to treat the imatinib-resistant leukemia, which are also resistant to TRAIL.

Superoxide activates mTOR-eIF4E-Bax route to induce enhanced apoptosis in leukemic cells.

Mammalian target of rapamycin (mTOR) is a central kinase that regulates cell survival, proliferation and translation. Reactive oxygen species (ROS) are second messengers with potential in manipulating cellular signaling. Here we report that two ROS generating phytochemicals, hydroxychavicol and curcumin synergize in leukemic cells in inducing enhanced apoptosis by independently activating both mitogen activated protein kinase (MAPK) (JNK and P(38)) and mTOR pathways. Low level transient ROS generated after co-treatment with these phytochemicals led to activation of these two pathways. Both mTOR and MAPK pathways played important roles in co-treatment-induced apoptosis, by knocking down either mTOR or MAPKs inhibited apoptosis. Activation of mTOR, as evident from phosphorylation of its downstream effector eukaryotic translation initiation factor 4E-binding protein 1, led to release of eukaryotic translation initiation factor 4E (eIF4E) which was subsequently phosphorylated by JNK leading to translation of pro-apoptotic proteins Bax and Bad without affecting the expression of anti-apoptotic protein Bcl-xl. Our data suggest that mTOR and MAPK pathways converge at eIF4E in co-treatment-induced enhanced apoptosis and provide mechanistic insight for the role of mTOR activation in apoptosis.

Orchestration of immune response by innate lymphoid cell subtype 2 at various tumor microenvironment, a suitable target for cancer immunotherapy.

Innate lymphoid cells are a mixed population of cells and critical regulators of our innate immune system. According to recent scientific literature, tissue resident innate lymphoid cell subtype 2 has been recognized as an important player of type 2 inflammatory responses, involved in different human malignancies like pancreatic, lung, acute myeloid leukemia, gastrointestinal tract cancer, etc. The current reports have revealed that, among the three main ILC sub types, subtype 2 (ILC 2), as the key regulator of initiating the type 2 inflammatory responses at the tumor microenvironment (TME). This activation of ILC-2 is a very important step for the specific downstream functioning of ILC-2. Priming of ILC-2 with different chemokines involves different cytokine secretion from the activated ILC-2 like IL-4, IL-5, IL-13, IL-9 which induce type 2 inflammatory responses involved in the complex interaction with other immune cells like NK cell, Cytotoxic T cell, MDSC and Treg cell. At the initial stage, ILC-2 activation through IL-33 may induce the anti-tumorigenic effect mediated by ILC-2/eosinophil axis. However, it is also evident that PDG2 (Prostaglandin D2)-mediated activation of ILC-2 induces the ILC-2/MDSC immune suppressive pro-tumorigenic niche at the TME. Here, in this review, we have summarized the function of ILC-2 on cancer immunity based on recent scientific work which indicates ILC-2 plays a dual role and orchestrates the immune responses toward type 2 immunity in different cancer settings.

Cancer is a complex disease where abnormal growth of body cells destroys the normal functions of affected body tissue. It is also one of the leading causes of death worldwide across every continent. There are more than 100 human cancers have been identified until now. Our body has an immune system, which always fights against any harmful agent including cancer cells, which try to invade our immune system and makes us fall ill. Our immune system consists of different specialized cells termed as immune cells which may involve in the direct killing of the cancer cells or it may indirectly help other cells to do it via complex interactions. Innate lymphoid cell or ILC is such type of immune cells of our immune system. Innate lymphoid cell has three main subtypes ILC-1, ILC-2, ILC-3; however, in the context of cancer development and progression, recently, ILC-2 has been recognized as one of the key players. Thus, in this article, we have summarized the activation and various functions of ILC-2 in different cancer settings which conclude that, ILC-2 can be a reasonably good therapeutic target to combat the devastating disease called cancer.

Implications of xenobiotic-response element(s) and aryl hydrocarbon receptor in health and diseases.

The effect of air pollution on public health is severely detrimental. In humans; the physiological response against pollutants is mainly elicited via the activation of aryl hydrocarbon receptor (AhR). It acts as a prime sensor of xenobiotic chemicals, also functioning as a transcription factor regulating a variety of gene expressions. Along with AhR, another pivotal element of the pollution stress pathway is Xenobiotic Response Elements (XREs). XRE, as studied are some conserved sequences in the DNA, responsible for the physiological response against pollutants. XRE is present at the upstream of the inducible target genes of AhR and it regulates the function of the AhR. XRE(s) are highly conserved in species as it has only eight specific sequences found so far in humans, mice, and rats. Inhalation of toxicants like dioxins, gaseous industrial effluents, and smoke from burning fuel and tobacco leads to predominant damage to the lungs. However, scientists are exploring the involvement of AhR in chronic diseases for example chronic obstructive pulmonary disease (COPD) and also other lethal diseases like lung cancer. In this review, we summarise what is known at this time about the roles played by the XRE and AhR in our molecular systems that have a defined control in the normal maintenance of homeostasis as well as dysfunctions.

Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

ICB3E induces iNOS expression by ROS-dependent JNK and ERK activation for apoptosis of leukemic cells.

The role of c-Jun N terminal Kinase (JNK) has been well documented in various cellular stresses where it leads to cell death. Similarly, extracellular signal-regulated kinase (ERK) which was identified as a signalling molecule for survival pathway has been shown recently to be involved in apoptosis also. Recently we reported that ICB3E, a synthetic analogue of Piper betle leaf-derived apoptosis-inducing agent hydroxychavicol (HCH), possesses anti-chronic myeloid leukemia (CML) acitivity in vitro and in vivo without insight on mechanism of action. Here we report that ICB3E is three to four times more potent than HCH in inducing apoptosis of leukemic cells without having appreciable effects on normal human peripheral blood mononuclear cells, mouse fibroblast cell line NIH3T3 and monkey kidney epithelial cell line Vero. ICB3E causes early accumulation of mitochondria-derived reactive oxygen species (ROS) in K562 cells. Unlike HCH, ICB3E treatment caused ROS dependent activation of both JNK, ERK and induced the expression of iNOS leading to generation of nitric oxide (NO). This causes cleavage of caspase 9, 3 and PARP leading to apoptosis. Lack of cleavage of caspase 8 and inability of blocking chimera antibody to DR5 or neutralizing antibody to Fas to reverse ICB3E-mediated apoptosis suggest the involvement of only intrinsic pathway. Our data reveal a novel ROS-dependent JNK/ERK-mediated iNOS activation pathway which leads to NO mediated cell death by ICB3E.

Manipulation of Inflammasome: A Promising Approach Towards Immunotherapy of Lung Cancer.

Chronic inflammation has emerged as a key player at different stages of cancer development. A prominent signaling pathway for acute and chronic inflammation is the activation of the caspase-1 inflammasomes. These are complexes that assemble on activation of certain nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs), AIM2-like receptors (ALRs), or pyrin due to activation via PAMPs or DAMPs. Of these, five complexes-NLRP1, NLRP3, NLRC4, Pyrin, and AIM2 are of importance in the context of cancer for their activities in modulating immune responses, cell proliferation, and apoptosis. Inflammasomes have emerged as clinically relevant in multiple forms of cancer making them highly promising targets for cancer therapy. As lungs are a tissue niche that is prone to inflammation owing to its exposure to external substances, inflammasomes play a vital role in the development and pathogenesis of lung cancer. Therefore, manipulation of inflammasome by various immunomodulatory means could prove a full-proof strategy for the treatment of lung cancer. Here, in this review, we tried to explore the various strategies to target the inflammasomes for the treatment of lung cancer.

H2O2 mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562.

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

INTRODUCTION: Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H(2)O(2), which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. MATERIALS AND METHODS: Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. CONCLUSION: NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.

BACKGROUND: Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism. MATERIALS AND METHODS: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. RESULTS: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells. CONCLUSION/SIGNIFICANCE: BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl+ CML cells.

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl(+) cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl(+) cells.