A study on the anticancer activity of imidazolyl benzamide derivative-IMUEB on a 549 lung cancer cell line.

Background: Cancer is a deadly disease, which is due to the uncontrolled division of cells with abnormal or unusual characteristics. It is a consequence of lethal mutations occurring due to various chemical and physical carcinogens, affecting many cellular signalling pathways and leading to uncontrolled proliferation. In this study, we analyzed the effect of 4-(1H-imidazol-1-yl)-N-(2-(3-(4-methylbenzyl) ureido) ethyl)benzamide (IMUEB), an imidazole derivative, on A549 cells (lung cancer cells). Methods: The MTT and LDH assays were performed to measure the cytotoxicity of IMUEB against A549 cells. Apoptotic mode of cell death of A549 cells was determined by fluorescence imaging by using different stains. Flow cytometry was performed to detect the cell cycle arrest. Western blotting was performed to determine the levels of apoptotic protein. Wound healing assay was performed to find the effect of IMUEB on cell migration. In silico molecular docking of IMUEB was performed to predict its affinity towards apoptotic proteins and metastasis related enzymes. Result and Discussion: The MTT assay showed an increase in cytotoxicity with increasing concentrations of IMUEB. In addition, it was found that IMUEB arrests cell cycle at G1 phase as detected by flow cytometry analysis and induces apoptosis. The treatment with IMUEB drastically decreased the migratory potential of A549 cells as evaluated by migration and invasion assay. By Western blotting analysis, it was found that the concentration of caspase-3 was increased after the treatment with IMUEB. Conclusion: Altogether, our results indicate that IMUEB shows antitumor activity by inhibiting proliferation and inducing apoptosis in A549 cells.

Role of indole curcumin in the epigenetic activation of apoptosis and cell cycle regulating genes.

Background: Head-and-neck squamous cell carcinoma is associated with the epigenetic silencing of various genes such as DAPK, ataxia telangiectasia mutated (ATM), BRCA1, p16INK4a, pVHL, p16, and RASSF1A. The most common epigenetic change observed in these genes is DNA methylation that directs the studies toward finding inhibitors for DNA methyltransferases (DNMTs), the protagonist in the action. The present study focuses on analyzing the possibility whether indole curcumin can reverse epigenetic changes of the various tumor suppressor genes, characteristically silenced by methylation, by inhibiting the major methylation enzyme DNA methyltransferase 1 or DNMT1. Materials and Methods: The cytotoxic effects of indole curcumin were studied through the MTT and lactate dehydrogenase assays. To determine the apoptosis-mediated death of HEp-2 cells, fluorescence imaging using different stains was done. Gene or mRNA expression analysis was done for p53, ATM, and DAPK genes. Results: The results obtained from this study clearly indicate that the indole analog of curcumin plays a remarkable role in activating genes involved in cell cycle regulation and apoptosis induction through epigenetic regulation. The influence that the drug has on the methylation status of gene promoter sequence of the ATM gene is also very significant. Conclusion: Indole curcumin, being an analog of curcumin, promises to be a very useful drug molecule having various potential targets. The target selected for this study was DNMT1 enzyme and the drug seems to actually show the effects; it was predicted to be having on the target molecule.

Indole curcumin combats metastatic HBV-positive hepatocellular carcinoma by inhibiting cell proliferation, migration, and matrix metalloproteinase-9 activity.

Background: Chemical modification of the natural products and molecules can lead us toward drugs with lesser off-target effects for chemotherapeutic use against cancers. In this study, we explored the effect of an indole analog of the molecule curcumin, for the first time against HBV-positive hepatocellular carcinoma (HCC) cells in vitro. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase assays were used to measure the cytotoxic effects of indole curcumin against Hep3B cells. The mode of cell death was established through acridine orange/ethidium bromide fluorescence staining, propidium iodide fluorescence staining, and the comet assay. The effect of the compound on cell migration behavior was studied through wound healing assay, whereas the effect on matrix metalloproteinase (MMP) activity was evaluated using gelatin zymography. In silico molecular docking was performed to predict the affinity of indole curcumin toward probable intracellular interacting partners. Results and Discussion: Indole curcumin had an antiproliferative effect on Hep3B cells, induced apoptotic mode of cell death, inhibited cell migration in time- and dose-dependent assays, and decreased MMP-9 activity levels. Molecular docking results suggest that the interaction of PI3K with indole curcumin may have led to downregulation of MMP-9 expression, thereby contributing to the overall reduction in MMP-9 activity. Conclusion: Our study establishes that indole curcumin is an effective cytotoxic and antimetastatic agent against hepatitis virus-B positive HCC cells. Hence, it can be a possible candidate for the treatment of hepatocarcinoma induced/promoted by the presence of chronic hepatitis B infection.

Epigenetic modulation and apoptotic induction by a novel imidazo-benzamide derivative in human lung adenocarcinoma cells.

PURPOSE: Lung cancer is the most commonly diagnosed and leading cause of cancer death worldwide. Imidazo-benzamides are considered to be good anti-cancer agents. The present study was aimed to investigate the cytotoxicity of a novel imidazo-benzamide derivative N-(2-(3-(tert-butyl)ureido)ethyl)-4-(1H-imidazol-1-yl)benzamide (TBUEIB) in lung cancer cell line A549. METHODS: The antiproliferative activity of TBUEIB was investigated using MTT, LDH and trypan blue assay. The apoptotic potential was investigated using various staining techniques and further confirmed by DNA fragmentation assay and western blotting. RESULTS: TBUEIB inhibited fifty precent A549 cells at a dose of 106 µM. The novel compound was found to exert a modulatory effect on apoptotic marker caspase-3 as well as epigenetic regulatory proteins like DNA Methyltransferase 1 (DNMT1). In silico studies with the compound and other epigenetic proteins such as Histone deacetylase (HDAC) and ubiquitin-like with PHD (plant homeodomain) and RING (Really Interesting New Gene) finger domains 1(UHRF1) showed good modulatory effects. CONCLUSION: The overall results obtained in the study conclude that the novel compound TBUEIB has potential anti-cancer activities, mainly by targeting the expression of DNMT1 enzyme, which may have re-activated the major tumor suppressor genes involved in the cell cycle, leading to the apoptosis of the cancer cells. The results also indicate that the compound has more than one target in the epigenetic pathway implying that the compound may be a potential multi-target compound.

Synthetic curcumin analog: inhibiting the invasion, angiogenesis, and metastasis in human laryngeal carcinoma cells via NF-kB pathway.

BACKGROUND: Laryngeal carcinoma, the most common among head and neck squamous cell carcinoma (HNSCC), induces 1% of all cancer deaths. Curcumin the active constituent of turmeric, is shown to be effective in the treatment of various cancers. In the present study, we explored the mechanistic role of bis-demethoxy curcumin analog (BDMC-A) as a chemotherapeutic agent. We investigated its inhibitory effect on invasion, angiogenesis, and metastasis in human laryngeal carcinoma (Hep-2) cells in comparison with curcumin. METHODS: The effect of curcumin and BDMC-A on transcription factors (NF-κB, p65, c-Jun, c-Fos, STAT3, 5, PPAR-γ, ß-catenin, COX-2, MMP-9, VEGF, TIMP-2) involved in signal transduction cascade, invasion, and angiogenesis in Hep-2 cells were quantified using Western blotting and RT-PCR technique. ELISA was used to measure the pro-inflammatory markers in Hep-2 cells treated with curcumin and BDMC-A. RESULTS: The results showed that BDMC-A inhibits the transcription factors NF-κB, p65, c-Jun, c-Fos, STAT3, STAT5, PPAR-γ and ß-catenin, which are responsible for tumor progression and malignancy. Moreover, BDMC-A treatment downregulated MMP-9, VEGF, TGF- ß, IL-6 and IL-8 and upregulated TIMP-2 levels. The effects were more significant compared to curcumin. CONCLUSION: Our overall results revealed that BDMC-A more effectively inhibited the markers of invasion, angiogenesis and metastasis in comparison with curcumin.

A novel anticancer chromeno-pyrimidine analogue inhibits epithelial-mesenchymal transition in lung adenocarcinoma cells.

Cancer is the second most dreaded disease worldwide. It is either acquired or inherited leading to the accompanying undesirable changes in the affected cells. Most existing chemotherapeutic drugs show enormous side effects. To minimize such effects, constant progress has been observed in the field of cancer by screening the anti-cancer effects of different chemical analogues. In the current study, we investigated the mechanism of action of a novel anticancer chromeno-pyrimidine analogue. We employed MTT, LDH assay to study cytotoxicity. DNA fragmentation, fluorescence imaging, and flow cytometric techniques have been carried out to study apoptosis, ROS generation, and cell cycle respectively. Wound healing assay and western blotting were used to evaluate the markers of epithelial-mesenchymal transition associated with metastasis. Molecular docking was used to predict possible protein targets that bind to this compound. The novel analogue induced apoptosis in lung adenocarcinoma cells and exhibited anti-metastatic activity. Increased expression of E-cadherin and inhibition of epithelial-mesenchymal transition was also observed. Docking studies with metastasis-related proteins such as Frizzled-7 (CRD), and Snail1 predict a high binding affinity of CP4b to both proteins. The novel analogue is therefore an anti-metastatic compound with EMT-inhibiting property and is hypothesized to act via binding to multiple targets in cancer cells.

Enhanced drug retention, sustained release, and anti-cancer potential of curcumin and indole-curcumin analog-loaded polysorbate 80-stabilizied PLGA nanoparticles in colon cancer cell line SW480.

The major therapeutic limitation of curcumin and indole-incorporated curcumin analog is its low bioavailability. We hypothesized that nano-encapsulation of indole-incorporated curcumin analog and curcumin as a biodegradable polymeric nanoparticle may enhance its bioavailability with extended drug retention time. Indole-incorporated curcumin analog and curcumin loaded PLGA nanoparticles were synthesized by solvent evaporation technique. Physicochemical characterizations and anti-cancer potential of the nanoparticles were evaluated in human colon cancer cell line SW480. The synthesized NPs had a size range of 50-150 nm diameter. The nano-formulation preserved the drug from degradation in wide ranges of pH environments. The nanoparticles treatment against SW480 cancer cell line triggered nuclear fragmentation, cell cycle blockade, inhibition of apoptosis and metastatic biomarkers. These drug-loaded nanoparticles may be potent nano-formulations against colon cancer because of its ability to tolerate extreme pH environments, thus having potential of oral drug-delivery.

Redox Nano-Architectures: Perspectives and Implications in Diagnosis and Treatment of Human Diseases.

SIGNIFICANCE: Efficient targeted therapy with minimal side-effects is the need of the hour. Locally altered redox state is observed in several human ailments, such as inflammation, sepsis, and cancer. This has been taken advantage of in designing redox-responsive nanodrug carriers. Redox-responsive nanosystems open a door to a multitude of possibilities for the control of diseases over other drug delivery systems. Recent Advances: The first-generation nanotherapy relies on novel properties of nanomaterials to shield the drug and deliver it to the diseased tissue or organ. The second generation is based on targeting the drug or diagnostic material to the diseased cell-specific receptors, or to a particular organ to improve the efficacy of the drug. The third and the latest generation of nanocarriers, the stimuli-responsive nanocarriers exploit the disease condition or environment to specifically deliver the drug or diagnostic probe for the best diagnosis and treatment. Several different kinds of stimuli such as temperature, magnetic field, pH, and altered redox state-responsive nanosystems have educed immense promise in the field of nanomedicine and therapy. CRITICAL ISSUES: We describe the evolution of nanomaterial since its inception with an emphasis on stimuli-responsive nanocarriers, especially redox-sensitive nanocarriers. Importantly, we discuss the future perspectives of redox-responsive nanocarriers and their implications. FUTURE DIRECTIONS: Redox-responsive nanocarriers achieve a near-to-zero premature release of the drug, thus avoiding off-site toxicity associated with the free drug. This bears great potential for the development of more effective drug delivery with better pharmacokinetics and pharmacodynamics.

Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans-activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans-activation response (TAR) RNA. In this study, HIV-1 infected patients (n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

Identification of natural inhibitors of Bcr-Abl for the treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cells, characterized at the molecular level by the bcr/abl gene rearrangement. Even though targeting the fusion gene product Bcr-Abl protein is a successful strategy, development of drug resistance and that of drug intolerance are currently the limitations for Bcr-Abl-targeted CML therapy. With an aim to develop natural Bcr-Abl inhibitors, we performed virtual screening (VS) of ZINC natural compound database by docking with Abl kinase using Glide software. Two natural inhibitors ZINC08764498 (hit1) and ZINC12891610 (hit2) were selected by considering their high Glide docking score and critical interaction with the hinge region residue Met-318 of Abl kinase. The reactivity of the two molecules was assessed computationally by density functional theory calculations. Further, the conformational transition, hydrogen bond interactions, and the binding energies were investigated during 10-ns molecular dynamics simulation of the Abl-hit complex. When tested in vitro, hit1 compared to hit2 showed selective inhibition of cell proliferation and induction of apoptosis in Bcr-Abl-positive K-562 leukemia cells. In summary, our results demonstrate that ZINC08764498, a coumarin derivative identified through VS, is a potential natural inhibitor for the treatment of CML.

Anti-proliferative and apoptosis-triggering potential of disulfiram and disulfiram-loaded polysorbate 80-stabilized PLGA nanoparticles on hepatocellular carcinoma Hep3B cell line.

There is an emerging trend to restudy known drugs for their anti-cancer potential. One such anti-alcoholic drug, disulfiram, with significant anti-cancer potential was studied for its efficacy against Hep3B cell lines, an in vitro model of hepatocellular carcinoma. Simultaneously, we intended to study the effect of polysorbate 80-stabilized PLGA nanoparticles and its DSF-loaded counterpart. Cell and nuclear staining, comet assay, flow cytometry and Western blots were performed. Results suggest that cell proliferation was inhibited by DSF and its PLGA nanoparticles through cell cycle arrest, triggering activation of apoptotic pathways that culminates with cell death. DSF loaded nanoparticles when compared with free DSF, showed significantly lesser effect due to its sustained drug-releasing property, while empty nanoparticles showed negligible influence on Hep3B cells. Our results suggest that DSF alone contributes to cell death, while polysorbate 80-stabilized PLGA nanoparticles show sustained drug release patterns that would potentially lower dosage regimens.

Hypoglycaemic role of wheatgrass and its effect on carbohydrate metabolic enzymes in type II diabetic rats.

Diabetes mellitus (DM) is a leading cause of morbidity and mortality in the world. Insulin resistance and insulin insufficiency is the major factor for the prognosis of type II diabetes. Consistent high glucose level leads to multiple secondary complications in diabetic patients. Hence, hypoglycaemic drugs are of significance for reducing the risk of secondary complications in type II diabetes. Various hypoglycaemic drugs are already available in the market, but they are associated with several side effects. Therefore, traditional herbs have emerged as safer alternative for effective hypoglycaemic treatment. The juvenile grass of common wheat is known as wheatgrass (WG). It is commonly used as a health drink and has potent antioxidant efficacy. It has been used to cure DM in folk medicine. The current study was planned to test the hypoglycaemic effect and pathways regulated by WG on DM. We analysed the glucose and insulin levels in plasma, the activity of glucose oxidative enzymes, hexokinase and glucose 6 phosphate dehydrogenase, in serum and glycogen levels in liver of the male albino Wistar rats. Activity of glucose oxidative enzymes and the levels of insulin and liver glycogen were decreased in rats with diabetes, but they were reversed on treatment with WG. Hence, we conclude that WG can act as a potent anti-hyperglycaemic agent.

BDMC-A, an analog of curcumin, inhibits markers of invasion, angiogenesis, and metastasis in breast cancer cells via NF-κB pathway--A comparative study with curcumin.

Breast cancer chemoprevention has become increasingly important in India as it faces a potential breast cancer epidemic over the next decade. Curcumin, the active ingredient in turmeric is a well known chemopreventive agent that possesses various therapeutic properties including antioxidants and anti-inflammatory effects. In the present study, we have investigated the inhibitory effects of BDMC-A, an analog of curcumin, on invasion, angiogenesis and metastasis markers using in vitro with MCF-7 cells and in silico studies, hence proved that BDMC-A has more potential than curcumin. Mechanistic studies revealed that BDMC-A might have exerted its activity by inhibiting metastatic and angiogenic pathways by modulating the expression of proteins upstream to NF-κB (TGF-ß, TNF-α, IL-1ß and c-Src), and NF-κB signaling cascade (c-Rel, COX-2, MMP-9, VEGF, IL-8) and by upregulating TIMP-2 levels. An in silico molecular docking study with NF-κB revealed that the docking score and interaction of BDMC-A with NF-κB-DNA binding was more efficient when compared to curcumin. Our overall results showed that BDMC-A more effectively inhibited invasion, angiogenesis and metastasis markers compared to curcumin. The activity can be attributed to the presence of hydroxyl group in the ortho position in its structure. Further research are going on to prove its potential as a therapeutic agent for breast cancer.

Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways.

BACKGROUND: Head and neck cancer is the sixth most frequently occurring cancer worldwide and accounts for about 2% of all cancer-related deaths annually. Curcumin is a well-known chemopreventive agent, and apoptosis induction by curcumin has been reported in many cancer cell types. We synthesized an ortho-hydroxy substituted analog of curcumin, bisdemethoxycurcumin analog (BDMC-A), and aimed to demarcate the apoptotic effects induced by BDMC-A on human laryngeal cancer Hep-2 cells and to compare these effects with those induced by curcumin. METHODS: We evaluated the apoptotic effects of BDMC-A in comparison to those of curcumin on Hep-2 cells by performing Western blotting, RT-PCR, fluorescent staining and DNA fragmentation assays. In addition, we carried out an in silico molecular docking study on the EGFR kinase domain. RESULTS: We found that BDMC-A can induce apoptosis in Hep-2 cells by regulating the expression of both intrinsic and extrinsic apoptotic proteins, i.e., Bcl-2, Bax, apoptososme complex and death receptors, more efficiently than curcumin. We also observed increased nuclear fragmentation and chromatin condensation after BDMC-A treatment compared to curcumin treatment. Depolarized mitochondria and ROS generation was well pronounced in both BDMC-A and curcumin treated Hep-2 cells. Our in silico molecular docking study on the EGFR kinase domain revealed that BDMC-A may dock more efficiently than curcumin. CONCLUSIONS: From our results we conclude that BDMC-A can induce apoptosis in Hep-2 laryngeal carcinoma cells more effectively than curcumin, and that this activity can be attributed to the presence of a hydroxyl group at the ortho position within this compound.

Antiproliferative and apoptotic effects of Sesbania grandiflora leaves in human cancer cells.

Natural phytochemicals and their derivatives are good drug candidates for anticancer therapeutic approaches against multiple targets. We report here the initial findings from our studies on the anticancer properties of the leaves of the medicinal plant Sesbania grandiflora. In the current study, five different solvent fractions from the leaves of S. grandiflora were tested on cancer cell lines such as MCF-7, HepG2, Hep-2, HCT-15, and A549. The methanolic fraction of S. grandiflora was found to exert potent antiproliferative effects especially in the human lung cancer cell line, A549. Caspase 3 was activated in the methanolic fraction treated A549 cells thereby leading to cell death by apoptosis. DAPI staining, DNA laddering, and decrease in mitochondrial membrane potential further confirmed the apoptotic mode of cell death. The high levels of ROS intermediates as evidenced by DCF-DA staining could have played a role in the apoptotic induction. Decrease in levels of cyclin D1 and decrease in the activation of NFkB were observed in A549 cells on treatment with methanolic fraction, giving a hint on the possible mechanism of action. These results prove that the medicinal plant S. grandiflora can be explored further for promising candidate molecules to combat cancer, especially lung cancer.

Effect of wheatgrass on membrane fatty acid composition during hepatotoxicity induced by alcohol and heated PUFA.

Alcoholism is a broad term used for problems related to alcohol, medically considered as disease, specifically an addictive illness, abuse, and dependence. It is the major cause of liver disease in western countries. Alcoholic liver disease encompasses the hepatic alterations leading to fatty liver, hepatitis, and fibrosis or cirrhosis. Fried food items prepared with repeatedly heated polyunsaturated fatty acid (PUFA) exacerbate the disturbances induced by alcohol. The use of herbs to treat diseases is almost universal. Wheatgrass (WG) is used as a supplemental nutrition because of its unique curative properties. As it has antioxidant property, it prevents cancer, diabetes, and acts as liver cleanser. The present study was undertaken to evaluate the efficacy of WG on preserving membrane integrity in liver damage induced by alcohol and heated PUFA (ΔPUFA).The rats were divided into four groups. The animals in group 1 served as normal (standard diet), group 2 served as hepatotoxic (alcohol + ΔPUFA), group 3 served as treated (alcohol + ΔPUFA + WG), and group 4 served as WG control. The compositions of membrane fatty acid, total phospholipids, phospholipase A, C (PLA and PLC) were analyzed in liver to evaluate the effects of WG. Changes in fatty acid composition, decrease in phospholipids levels, and increase in PLA, PLC were observed in the diseased group. Restoration effect was seen in WG-treated rats. Histopathological observations were in correlation with the biochemical parameters. From the results obtained, we conclude that WG effectively protects the liver against alcohol and ΔPUFA-induced changes in fatty acid composition and preserves membrane integrity.

Mechanism of apoptotic induction in human breast cancer cell, MCF-7, by an analog of curcumin in comparison with curcumin--an in vitro and in silico approach.

In developing countries, survival rates for breast cancer are poor and it accounts for 22.9% of all cancers in women. Curcumin, a major constituent from turmeric, is one of the well-known chemopreventive agents. Reports have shown that curcumin induces apoptosis in breast cancer cells. We synthesized an ortho-hydroxy substituted analog of curcumin (BDMC-A) and analyzed its cytotoxicity. The BDMC-A inhibited MCF-7 at a dose equivalent to that of curcumin (30 µM). The present study was aimed at delineating the apoptotic mechanism of BDMC-A in comparison to that of curcumin. In our study, BDMC-A exerted more potent effect on the modulation of selective apoptotic markers (intrinsic pathway: p53, Bcl-2, Bax, cyt c, Apaf-1, caspase-9, 3, PARP; extrinsic pathway: FasL, caspase 8) compared to curcumin. mRNA expression studies for Bcl2/Bax also supported the increased efficacy of BDMC-A. An in silico molecular docking study with PI3K revealed that the docking of BDMC-A was more potent compared to curcumin. Increased apoptotic induction by BDMC-A compared to curcumin was also demonstrated by Annexin V, Rh123 (ΔΨm), PI, Hoechst 33258, AO/EB fluorescent staining studies which showed characteristic apoptotic features like nuclear fragmentation and chromatin condensation. Moreover, BDMC-A treated cells effectively induced apoptosis through ROS intermediates compared to curcumin, as measured by 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA). Hence our overall results showed that BDMC-A induced apoptosis more effectively compared to curcumin and the activity can be attributed to the presence of hydroxyl group in the ortho position in its structure. Further researches are going on to delineate its molecular targets to evaluate its effect as a potent anticancer agent.