Benzimidazole-linked pyrazolo[1,5-a]pyrimidine conjugates: synthesis and detail evaluation as potential anticancer agents.

A library of benzimidazole briged pyrazolo[1,5-a]pyrimidine (6a-q) was designed, synthesized and subjected for evaluation for cytotoxic potential. Antiproliferative activity, ranging from 3.1-51.5 µM, was observed against a panel of cancer cell lines which included MCF-7 (breast cancer), A549 (lung cancer), HeLa (cervical cancer) and SiHa (cervical cancer). Among them, 6k, 6l, 6n and 6o have shown significant cytotoxicity and were investigated further to study their probable mechanism of action against MCF-7 cell line. Accumulation of cells at sub-G1 phase was observed in flow cytometric analysis. The detachment of cells from substratum and membrane blebbing seen under bright field microscopy supports the ability of these conjugates to induce apoptosis. Immunostaining and western blot analysis showed EGFR, p-EGFR, STAT3, and p-STAT3 significant downregulation. Western blot analysis demonstrated an elevated level of apoptotic proteins such as p53, p21, Bax, whereas a decrease in the antiapoptotic protein Bcl-2 and procaspase-9, confirming the ability of these conjugates to trigger cell death by apoptosis. EGFR kinase assay confirms the specific activity of conjugates. Molecular docking simulation study disclosed that these molecules fit well in ATP-binding pocket of EGFR. The analysis of docking poses and the atomic interactions of different conjugates rationalize the structural-activity relationship in this series. Benzimidazole-linked pyrazolo[1,5-a]pyrimidine conjugates were synthesized and evaluated for their anticancer potential. All the conjugates have significant anticancer potential. Further mechanistic studies revealed that these conjugates arrest cancer cell growth by EGFR/STAT3 inhibition.

Synthesis, 18F-radiolabeling and apoptosis inducing studies of novel 4, 7-disubstituted coumarins.

In present study, a new series of 4, 7-disubstituted coumarin derivatives (7a-y) have been synthesized as galectin-1 targeting apoptosis inducing agents and evaluated for their in vitro cytotoxic potentials against a panel of selected human cancer cell lines namely, Brest (MCF7), Ovarian (SKOV3), Prostate (PC-3 & DU145) and normal embryonic kidney (HEK293T) cells, using MTT assay. Most of the compounds exhibited potent growth inhibitory action against the treated cancer cell lines with an IC50 range of 10-30 µM. Compound 7q exhibited a significant growth inhibition against prostate cancer (PC-3 & DU145) cell lines with an IC50 value of 7.45 ± 0.03 µM, 8.95 ± 0.17 µM respectively. Further, the target compound 7q was radiolabeled with fluorine-18 [18F] to be used as a novel PET radiotracer for imaging of tumors via targeting galectin-1, using appropriate reaction conditions in the GE Tracer-lab FX2N synthesis module. The purification of the [18F] radiolabeled compound [18F]-7q was successfully achieved with 60% ethanol. The radiochemical purity was>85% and residual solvent limits of DMF was 65 ± 3 ppm as analysed by HPLC, TLC & GC analytical methods. The apoptosis studies confirm the inhibition of cell proliferation with morphological changes like cell shrinkage, blebbing and cell wall deformation, increasing the ROS levels, and loss of mitochondrial membrane potential by Acridine orange/Ethidium bromide staining, Hoechst-33342 staining, H2DCFDA staining, annexin V-FITC/PI, and JC-1 staining methods. In flow cytometric analysis, 7q selectively arrested the sub-G1 phase of the cell cycle in a dose-dependent manner. In Gal-1 ELISA studies, compound 7q efficiently reduced the levels of Gal-1 protein in dose-dependent manner with an IC50 value of 100 µM. The binding constant (Ka) of 7q with Gal-1 was observed as 1.3 × 104 M-1 by fluorescence spectroscopy. The molecular docking studies clearly showed possible interactions and the pharmacokinetic (ADMET) properties of compound 7q with Gal-1. Hence, the novel 4, 7-disubstituted coumarins could be a potential cytotoxic and PET imaging agents via Gal-1.

Insulin/insulin-like growth factor-1 signalling (IIS) based regulation of lifespan across species.

An organism's well-being is facilitated by numerous molecular and biochemical pathways that ensure homeostasis within cells and tissues. Aging causes a gradual let-down in the maintenance of homeostasis due to various endogenous and environmental challenges, leading to amassing of damages, functional deterioration of different tissues and vulnerability to ailments. Nutrient sensing pathways that maintain glucose homeostasis in body are involved in regulation of aging. Insulin/insulin-like growth factor-1 (IGF-1) signalling (IIS) pathway was the first nutrient sensing pathway discovered to affect the aging process. This pathway is highly conserved and the most studied among different organisms. Epigenetic machineries that include DNA and histone modifying enzymes and various non-coding RNAs have been identified as important contributors to nutrition-related longevity and aging control. In this report, we present the homology and differences in IIS pathway of various organisms including worm, fly, rodent and human. We also discuss how epigenome remodelling, chromatin based strategies, small and long non-coding RNA are involved to regulate multiple steps of aging or age-related insulin homeostasis. Enhanced study of the role of IIS pathway and epigenetic mechanisms that regulate aging may facilitate progressive prevention and treatment of human age-related diseases.

Long non-coding RNA: Functional agent for disease traits.

In recent years, long non-coding RNAs (lncRNAs) have attracted the attention of researchers with their involvement in all facets of life. LncRNAs are transcripts of more than 200 nucleotides which lack defined protein coding potential. Although they do not code for proteins, a large number of them are involved in regulating gene expression and translation. The presence of numerous lncRNAs in the human genome has prompted us to investigate the contribution of these molecules to human biology and medicine. In this review, we present the potential role of lncRNAs interlinked to different human diseases and genetic disorders. We also describe their role in cellular differentiation and aging and discuss their potential importance as biomarkers and as therapeutic agents.

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

Pigmy MicroRNA: surveillance cops in Therapies kingdom.

MicroRNAs (miRNAs) are well preserved in every animal. These pigmy sized non-coding RNAs (21-23 nt), scattered in genome, are responsible for micromanaging the versatile gene regulations. Involvement of miRNAs was surveillance cops in all human diseases including cardiovascular defects, tumor formation, reproductive pathways, and neurological and autoimmune disorders. The effective functional role of miRNA can be reduced by chemical entities of antisense oligonucleotides and versatile small molecules that support the views of novel therapy of different human diseases. In this study, we have updated our current understanding for designing and synthesizing miRNA-controlling therapeutic chemicals. We have also proposed various in-vivo delivery strategies and their ongoing challenges to combat the incorporation hurdles in live cells and animals. Lastly, we have demonstrated the current progress of miRNA modulation in the treatment of different human diseases that provides an alternative approach of gene therapy.

Synthesis and mechanistic aspects of 2-anilinonicotinyl-pyrazolo[1,5-a]pyrimidine conjugates that regulate cell proliferation in MCF-7 cells via estrogen signaling.

A series of anilinonicotinyl linked pyrazolo[1,5-a]pyrimidine conjugates (6a-x) were synthesized and evaluated for their antiproliferative activity. Some of these conjugates exhibited promising cytotoxic effects in the MCF-7 cell line and among these 6a and 6c exhibited significant effects, apart from G2/M cell cycle arrest. Interestingly they showed profound effects on cyclin D1, Bcl-2 and survivin proteins that regulate breast cancer cell proliferation. Moreover, ER alpha protein expression was studied to understand regulatory role of these conjugates on estrogen activity in estrogen positive breast cancer cells like MCF-7 and compounds 6a and 6c reduced their activity.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Computational approach for elucidating interactions of cross-species miRNAs and their targets in Flaviviruses.

BACKGROUND & OBJECTIVES: Combating viral diseases has been a challenging task since time immemorial. Available molecular approaches are limited and not much effective for this daunting task. MicroRNA based therapies have shown promise in recent times. MicroRNAs are tiny non-coding RNAs that regulate translational repression of target mRNA in highly specific manner. METHODS: In this study, we have determined the target regions for human and viral microRNAs in the conserved genomic regions of selected viruses of Flaviviridae family using miRanda and performed a comparative target selectivity analysis among them. RESULTS: Specific target regions were determined and they were compared extensively among themselves by exploring their position to determine the vicinity. Based on the multiplicity and cooperativity analysis, interaction maps were developed manually to represent the interactions between top-ranking miRNAs and genomes of the viruses considered in this study. Self-organizing map (SOM) was used to cluster the best-ranked microRNAs based on the vital physicochemical properties. INTERPRETATION & CONCLUSION: This study will provide deep insight into the interrelation of the viral and human microRNAs interactions with the selected Flaviviridae genomes and will help to identify cross-species microRNA targets on the viral genome.

Tandem Prins/pinacol reaction for the synthesis of oxaspiro[4.5]decan-1-one scaffolds.

A novel Lewis acid catalyzed Prins/pinacol cascade process has been developed for the synthesis of 7-substituted-8-oxaspiro[4.5]decan-1-ones in good yields with excellent selectivity. This is the first example of the synthesis of oxaspirocycles from aldehydes and 1-(4-hydroxybut-1-en-2-yl)cyclobutanol through a cascade of Prins/pinacol rearrangement. This method is applicable to a wide range of aldehydes such as aromatic, aliphatic, heteroaromatic, and α,ß-unsaturated aldehydes.

Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

BACKGROUND: In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. RESULTS: Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. CONCLUSION: mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.

Synthesis and study of benzothiazole conjugates in the control of cell proliferation by modulating Ras/MEK/ERK-dependent pathway in MCF-7 cells.

By applying a methodology, a series of benzothiazole-pyrrole based conjugates (4a-r) were synthesized and evaluated for their antiproliferative activity. Compounds such as 4a, 4c, 4e, 4g-j, 4m, 4n, 4o and 4r exhibited significant cytotoxic effect in the MCF-7 cell line. Cell cycle effects were examined for these conjugates at 2 µM as well as 4 µM concentrations and FACS analysis show an increase of G2/M phase cells with concomitant decrease of G1 phase cells thereby indicating G2/M cell cycle arrest by them. Interestingly 4o and 4r are effective in causing apoptosis in MCF-7 cells. Moreover, 4o showed down regulation of oncogenic expression of Ras and its downstream effector molecules such as MEK1, ERK1/2, p38 MAPK and VEGF. The apoptotic aspect of this conjugate is further evidenced by increased expression of caspase-9 in MCF-7 cells. Hence these small molecules have the potential to control both the cell proliferation as well as the invasion process in the highly malignant breast cancers.

Novel anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates modulate the expression of p53-MYCN associated micro RNAs in neuroblastoma cells and cause cell cycle arrest and apoptosis.

It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics.

Quinazolino linked 4ß-amidopodophyllotoxin conjugates regulate angiogenic pathway and control breast cancer cell proliferation.

A series of new conjugates of quinazolino linked 4ß-amidopodophyllotoxins 10aa-af and 10ba-bf were synthesized and evaluated for their anticancer activity against human pancreatic carcinoma (Panc-1) as well as breast cancer cell lines such as MCF-7 and MDA-MB-231 by employing MTT assay. Among these conjugates, some of them like 10bc, 10bd, 10be and 10bf exhibited high potency of cytotoxicity. Flow cytometric analysis showed that these conjugates arrested the cell cycle in the G2/M phase and caused the increase in expression of p53 and cyclin B1 protein with concomitant decrease in Cdk1 thereby suggesting the inhibitory action of these conjugates on mitosis. Interestingly, we observed a decrease in expression of proteins that control the tumor micro environment such as VEGF-A, STAT-3, ERK1/2, ERK-p, AKT-1 ser 473 phosphorylation in compounds treated breast cancer cells. Further, these effective conjugates have exhibited inhibitory action on integrin (αVßIII). Furthermore, the MCF-7 cells that were arrested and lost the proliferative capacity undergo mitochondrial mediated apoptosis by activation of caspases-9. Thus these conjugates have the potential to control breast cancer cell growth by effecting tumor angiogenesis and invasion.

4ß-[4'-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA topoisomerase I and IIα inhibitors and apoptosis inducing agents.

A series of 4ß-[4'-(1-(aryl)ureido)benzamide]podophyllotoxin congeners (11a-l) were synthesized and evaluated for their cytotoxic activity against six human cancer cell lines. Some of the compounds like 11a, 11h, 11k and 11l showed significant anti-proliferative activity in Colo-205 cells and were superior to etoposide. The flow-cytometric analysis studies indicated that these compounds show strong G1 cell cycle arrest, as well exhibited improved inhibitory activities on DNA topoisomerase I and IIα enzymes. These compounds induce apoptosis by up regulating caspase-3 protein as observed by ELISA and Western blotting analysis. In addition, a brief structure-activity relationship studies within the series along with docking results of representative compounds 11a, 11h, 11k, 11l were presented.

Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation.

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two "Argonaute" family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w-Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems.

Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells.

BACKGROUND: Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. METHODS: Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. RESULTS: Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. CONCLUSION: Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (-692 to -684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK inhibitor p21WAF1.

Synthesis and biological evaluation of novel triazoles and isoxazoles linked 2-phenyl benzothiazole as potential anticancer agents.

A new series of isoxazoles and triazoles linked 2-phenyl benzothiazole were synthesized and evaluated for their anticancer activity. These compounds have been tested for their cytotoxicity three cancer cell lines. Among the compounds tested, compound 5d showed good cytotoxicity against Colo-205 and A549 cells in comparison to standard control PMX 610(1). Further compound 5d has been tested for its apoptotic activity and its inhibitory activity against caspase and PARP proteins. Hence this compound has the potential that it can be selected for further biological studies.

Imidazo-benzothiazoles a potent microRNA modulator involved in cell proliferation.

MicroRNAs are endogenously expressed tiny non-coding RNAs that control gene expression at the post-transcriptional level and regulate processes of cell growth, differentiation, proliferation and apoptosis. Aberrant expression of microRNAs correlates with various cancers. Our experiments demonstrated that imidazo-benzothiazole conjugates caused apoptosis in colon cancer cells by modulating the expression of microRNAs. In vivo study in Drosophila melanogaster has exhibited inhibitory action on bantam microRNA, the homolog of human miR-542-5p that is involved in deciding the cellular cues that regulate the balance between proliferation and apoptosis. The expression of direct targets of bantam such as Hid and HDAC-6 were affected upon compound treatment. Interestingly, these conjugates downregulate the genes involved in microRNA biogenesis such as Drosha, Pasha and Dicer-1. Our findings have elucidated the microRNA inhibitory role of imidazo-benzothiazole conjugates.

Synthesis, anticancer activity and apoptosis inducing ability of bisindole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates.

A series of bisindole-pyrrolobenzodiazepine conjugates (5a-f) linked through different alkane spacers was prepared and evaluated for their anticancer activity. All compounds exhibited significant anticancer potency and the most potent compounds 5b and 5e were taken up for detailed studies on MCF-7 cell line. Cell cycle effects were examined apart from investigating the inhibition of tubulin polymerization for compounds 2a, 2b, 5b and 5e at 2µM. FACS analysis showed that at higher concentrations (4 and 8µM) there was an increase of sub-G1 phase cells and decrease of G2/M phase cells, thus indicating that compounds 5b and 5e are effective in causing apoptosis in MCF-7 cells. It was also observed that compounds 5b and 5e showed the down regulation of histone deacetylase protein levels such as HDAC1, 2, 3, 8 and increase in the levels of p21, followed by apoptotic cell death. The apoptotic nature of these compounds was further evidenced by increased expression of cleaved-PARP and active caspase-7 in MCF-7 cells.