Apoptosis-inducing Capability Research Articles

Pharmacological investigation of new niclosamide-based isatin hybrids as antiproliferative, antioxidant, and apoptosis inducers

A group of Niclosamide-linked isatin hybrids (Xo, X1, and X2) was created and examined using IR, 1HNMR, 13C NMR, and mass spectrometry. These hybrids' cytotoxicity, antioxidant, cell cycle analysis, and apoptosis-inducing capabilities were identified. Using the SRB assay, their cytotoxicity against the human HCT-116, MCF-7, and HEPG-2 cancer cell lines, as well as VERO (African Green Monkey Kidney), was evaluated. Compound X1 was the most effective compound. In HCT-116 cells, compound X1 produced cell cycle arrest in the G1 phase, promoted cell death, and induced apoptosis through mitochondrial membrane potential breakdown in comparison to niclosamide and the control. Niclosamide and compound X1 reduced reactive oxygen species generation and modulated the gene expression of BAX, Bcl-2, Bcl-xL, and PAR-4 in comparison to the control. Docking modeling indicated their probable binding modalities with the XIAP BIR2 domain, which selectively binds caspase-3/7, and highlighted their structural drivers of activity for further optimization investigations. Computational in silico modeling of the new hybrids revealed that they presented acceptable physicochemical values as well as drug-like characteristics, which may introduce them as drug-like candidates. The study proved that compound X1 might be a novel candidate for the development of anticancer agents as it presents antiproliferative activity mediated by apoptosis.

A Facile Synthesis and Molecular Characterization of Certain New Anti-Proliferative Indole-Based Chemical Entities.

Cancer cells frequently develop drug resistance, which leads to chemotherapeutic treatment failure. Additionally, chemotherapies are hindered by their high toxicity. Therefore, the development of new chemotherapeutic drugs with improved clinical outcomes and low toxicity is a major priority. Several indole derivatives exhibit distinctive anti-cancer mechanisms which have been associated with various molecular targets. In this study, target compounds 4a-q were obtained through the reaction of substituted benzyl chloride with hydrazine hydrate, which produces benzyl hydrazine. Subsequently, the appropriate substituted benzyl hydrazine was allowed to react with 1H-indole-2-carboxylic acid or 5-methoxy-1H-indole-2-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a coupling agent. All compounds exhibited cytotoxicity in three cell lines, namely, MCF-7, A549, and HCT. Compound 4e exhibited the highest cytotoxicity, with an average IC50 of 2 µM. Moreover, a flow cytometry study revealed a significantly increased prevalence of Annexin-V and 7-AAD positive cell populations. Several derivatives of 4a-q showed moderate to high cytotoxicity against the tested cell lines, with compound 4e having the highest cytotoxicity, indicating that it may possess potential apoptosis-inducing capabilities.

Induction of apoptosis by ethanolic extract of leaf of Dillenia pentagyna Roxb. in A549 cells via NF-κB pathway

BackgroundA large number of populations worldwide still rely on crude extracts of different parts of medicinal plants. Different parts of Dillenia pentagyna has long been used in India for the treatment of different disorders. ObjectiveCytotoxic and apoptosis-inducing capability of ethanolic extract of leaves of Dillenia pentagyna (EELDP) were studied on various human cancer cell lines. MethodsCell viability and morphology, nuclear fragmentation, caspase-3 activation, ratio of Bax/Bcl2, NF-κB expression were measured in three human cancer cells A549, HeLa and U2OS after treatment with EELDP. Further, mitochondrial membrane potential, cell cycle analysis, ROS generation were done in A549 cells. ResultsEELDP dose-dependently increased cell cytotoxicity to all the human cancer cells but showed nontoxic to the normal lung cell L-132. EELDP (0–0.6 mg/ml) significantly induced the intrinsic pathway of apoptosis via fall in mitochondrial membrane potential leading to an increase in Bax to Bcl2 ratio, the release of cytochrome-c, caspase-3 activation, and nuclear fragmentation. EELDP significantly reduced NF-κB expression. However, combined EELDP with Bay11–7082 (NF-κB inhibitor) treatment significantly enhanced Bax to Bcl2 ratio and caspase-3 activation. ConclusionThis data implicates that EELDP has a strong potential to kill various aggressive human cancer cells by inducing apoptosis via NF- κB pathway.

Assessment of Placental Extracellular Vesicles-Associated Fas Ligand and TNF-Related Apoptosis-Inducing Ligand in Pregnancies Complicated by Early and Late Onset Preeclampsia.

Preeclampsia (PE) is a hypertensive disorder that affects 2–8% of pregnancies and is one of the main causes of fetal, neonatal, and maternal mortality and morbidity worldwide. Although PE etiology and pathophysiology remain unknown, there is evidence that the hyperactivation of maternal immunity cells against placental cells triggers trophoblast cell apoptosis and death. It has also been reported that placenta-derived extracellular vesicles (EV) carry Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trigger apoptosis in Jurkat T cells. This study aimed to quantify and compare FasL and TRAIL expression in EV derived from cultures of placenta explants from women with PE (early versus late) and women with uncomplicated pregnancies. Also, the study assessed EV capacity to induce apoptosis in Jurkat T cells. The authors isolated EV from placenta explant cultures, quantified FasL and TRAIL using ELISA, and analyzed EV apoptosis-inducing capability by flow cytometry. Results showed increased FasL and TRAIL in EV derived from placenta of women with PE, and increased EV apoptosis-inducing capability in Jurkat T cells. These results offer supporting evidence that EV FasL and TRAIL play a role in the pathophysiology of PE.

Chemical Constituents of Eupatorium japonicum and Anti-Inflammatory, Cytotoxic, and Apoptotic Activities of Eupatoriopicrin on Cancer Stem Cells.

Eupatorium japonicum Thunb. of the plant family Asteraceae is a popular traditional herb in Vietnam. However, its chemical constituents as well as bioactive principles have not been investigated yet. We investigated the phytochemistry of E. japonicum in Vietnam and isolated seventeen compounds (1–17) including phytosterols, terpenoids, phenolic acids, flavonoids, fatty alcohols, and fatty acids. They were structurally determined by MS and NMR analysis. Except for compounds 6 and 12, all the other compounds were identified for the first time from E. japonicum. Since many sesquiterpene lactones with α-methylene γ-lactone ring are reported as anti-inflammatory and anticancer agents, eupatoriopicrin (10), 1-hydroxy-8-(4,5-dihydroxytigloyloxy)eudesma-4(15),11(13)-dien-6,12-olide (11) were selected among the isolates for biological assays. Compound 10 was identified as the main bioactive sesquiterpene lactone of E. japonicum showing its potent anti-inflammatory and cytotoxic activity through inhibiting NO production and the growth of HepG2 and MCF-7 human cancer cell lines. For the first time, eupatoriopicrin (10) was demonstrated to strongly inhibit NTERA-2 human cancer stem cell (CSC) line in vitro. It is noticeable that the cytotoxicity of eupatoriopicrin against NTERA-2 cells is mediated by its apoptosis-inducing capability of 10 as demonstrated by the results of Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays. The biological activities of the main bioactive constituents 1–7, 10, 12, and 15 supported the reported anti-inflammatory and anticancer properties of extracts from E. japonicum.

Synthesis, characterization and the anticancer activity of six lanthanides(III) complexes with 5,7-dihalogenated-8-quinolinol and 2,2’-bipyridine derivatives

Six new lanthanide(III) complexes, namely [GdIII(QL1)3(BL1)] (Gd1), [GdIII(QL1)3(BL2)] (Gd2), [LuIII(QL2)2(BL3)(NO3)] (Lu1), [LuIII(QL2)2(BL2)(NO3)] (Lu2), [YbIII(QL3)2(BL2)(NO3)] (Yb1), and [YbIII(QL4)2(BL2)(NO3)] (Yb2) coordinated with 5,7-dibromo-8-quinolinol (H-QL1), 5,5′-dimethyl-2,2′-bipyridyl (BL1), 4,4′-di-tert-butyl-2,2′-bipyridine (BL2), 5,7-dibromo-2-methylquinolin-8-ol (H-QL2), 2,2′-bipyridine (BL3), 5,7-dichloro-8-hydroxyquinoline (H-QL3), and 5,7-dichloro-8-hydroxy-2-methylquinoline (H-QL4), were prepared. The LnIII complexes Gd1, Gd2, Lu1, Lu2, Yb1 and Yb2 showed high cytotoxicity toward cervical (HeLa), ovarian (SK-OV-3) and lung (NCI-H460) cancer cells with IC50 values from 0.18 ± 0.14 to 8.02 ± 1.71 μM. In addition, Yb1 and Yb2 impaired mitochondrial functions, decreased mitochondrial membrane potential, and altered the levels of mitochondrial related-proteins. The apoptosis-inducing capability was in the order of Yb2 > Yb1. Such observed differences in biological activities could be caused by the electronic effect of the methyl group of H-QL4 in Yb2.

Synthesis of cucurbitacin IIa derivatives with apoptosis-inducing capabilities in human cancer cells.

Twenty-one cucurbitacin IIa derivatives were synthesized and screened for cytotoxic activity. Their structures were established using 1H NMR, 13C NMR, and LC-MS spectroscopic data. The absolute configuration of the derivatives was determined by single crystal diffraction. In sulforhodamine B (SRB) assays, nearly all compounds displayed low cytotoxicity toward normal human cells (HEK293). However, some derivatives displayed high cytotoxicity, in the low μM range, toward several human tumor cell lines (SKOV3, HT29, HEPG2, MCF-7, and LOVO). Low IC50 values were obtained, especially for acetyl-protected product 2, 2,4,6-trichlorophenylhydrazine derivative 4a, and 2-hydrazinopyridine derivative 4d. In particular, compounds 2 and 4d showed low IC50 values of 1.2 ± 0.01 and 2.2 ± 0.19 μM against SKOV3 cells. These compounds were submitted to extensive biological testing, which showed that compounds 2 and 4a did not inhibit tumor cells by influencing the cell cycle. Furthermore, compound 4a triggered the apoptotic pathway in cancer cells, showing high apoptosis ratios. This study mainly changed the structure of cucurbitacin tetracyclic triterpenoids and provided a novel tetracyclic skeleton derived from natural products that provided further references for the future modification of cucurbitacin tetracyclic triterpenoids.

Ficus extract-A promising agent for antimammary tumorigenesis: A review on current status and future possibilities.

Pharmacological studies have shown that various species of Ficus have antiviral, antidiarrheal, antipyretic, hypolipidemic, antidiabetic, antioxidant, anticancer, antiparasitic, antiangiogenic, anti-inflammatory, antibacterial, antiplatelet, reproductive, dermatological, immunological, endocrine, and hepato and nephron protective effects. But there is no sufficient research on biomolecules present in the leaf extract of Ficus religiosa and its mechanism of action. We have previously reported that bioavailable constituents of F.religiosa leaf extract exert photosensitizing and apoptosis-inducing capability through the generation of intracellular reactive oxygen species on breast cancer cells. In this review, we have evaluated the expression of checkpoint proteins of G1/S and sub G0 phase with wet lab data and also have done a data mining of other research for other potential mechanistic action of the F.religiosa leaf extract.

Mitochondrial dysfunction RAD51, and Ku80 proteolysis promote apoptotic effects of Dinaciclib in Bcl-xL silenced cells.

In the present study, we investigated the effect of CDK inhibitors (ribociclib, palbociclib, seliciclib, AZD5438, and dinaciclib) on malignant human glioma cells for cell viability, apoptosis, oxidative stress, and mitochondrial function using various assays. None of the CDK inhibitors induced cell death at a clinically relevant concentration. However, low nanomolar concentrations of dinaciclib showed higher cytotoxic activity against Bcl-xL silenced cells in a time- and concentration-dependent manner. This effect was not seen with other CDK inhibitors. The apoptosis-inducing capability of dinaciclib in Bcl-xL silenced cells was evidenced by cell shrinkage, mitochondrial dysfunction, DNA damage, and increased phosphatidylserine externalization. Dinaciclib was found to disrupt mitochondrial membrane potential, resulting in the release of cytochrome c, AIF, and smac/DIABLO into the cytoplasm. This was accompanied by the downregulation of cyclin-D1, D3, and total Rb. Dinaciclib caused cell cycle arrest in a time- and concentration-dependent manner and with accumulation of cells in the sub-G1 phase. Our results also revealed that dinaciclib, but not ribociclib or palbociclib or seliciclib or AZD5438 induced intrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bax and Bak), resulting in the activation of caspases and cleavage of PARP. We also found an additional mechanism for the dinaciclib-induced augmentation of apoptosis due to abrogation RAD51-cyclin D1 interaction, specifically proteolysis of the DNA repair proteins RAD51 and Ku80. Our results suggest that successfully interfering with Bcl-xL function may restore sensitivity to dinaciclib and could hold the promise for an effective combination therapeutic strategy.

Investigation of the cytotoxicity, apoptosis and pharmacokinetics of Raddeanin A.

Raddeanin A, one of the triterpenoid saponins extracted from Anemone raddeana rhizome of the Ranunculaceae family, has demonstrated the ability to inhibit the growth of human hepatic and gastric cancer cells. However, the effects of Raddeanin A on human colon cancer cells have not been investigated extensively. The present study aimed to examine the antiproliferative and apoptosis-inducing effects of Raddeanin A on the HCT-116 human colon cancer cell line in vitro, and evaluate the pharmacokinetic and biodistribution properties of Raddeanin A in mice following a single oral administration. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the in vitro cytotoxicity of Raddeanin A against HCT-116 cells. 4',6-Diamidino-2-phenylindole, dihydrochloride staining and flow cytometry were performed to further examine the apoptosis-inducing capability of Raddeanin A. The concentrations of Raddeanin A in the plasma and tissues were analyzed using liquid chromatography-tandem mass spectrometry. Raddeanin A showed a dose-dependent antiproliferative effect towards the HCT-116 cells, with a half maximal inhibitory concentration of ~1.4 µM. Treatment with Raddeanin A resulted in a significant induction of apoptosis, observed as apparent morphological changes of the nuclei, with a total apoptotic ratio of 41.8% at a concentration of 3 µM. Low concentrations of Raddeanin A were detected in the heart, liver, spleen, lung, kidney and plasma of the mice following oral administration, however, the majority of the Raddeanin A was distributed in the intestinal tract, particularly in the colon and caecum. These present study confirmed the growth-inhibitory and apoptosis-inducing effects of Raddeanin A on HCT-116 cells and performed preliminary examinations of its pharmacokinetic properties, which provide a foundation for further investigating the inhibitory mechanism on the colon cancer cells in vivo.

Highly efficient enzymatic synthesis of novel polydatin prodrugs with potential anticancer activity

Efficient lipase-mediated research work was successfully exploited for synthesizing potential 6″-O-acyl-polydatin prodrugs in biomass-derived 2-methyltetrahydrofuran (2-MeTHF). The results of the enzyme recognitions of nine acyl donors evidently demonstrated that the position and number of the CC double bond in acyl chains profoundly influenced the behavior of the enzyme, which could be attributable to the resonance effect between the double bond and carbonyl group. Further investigations showed that introducing various acyl groups into the polydatin apparently enhanced its pH stability and 1-octanol-water partition coefficient (log P). With regard to the human cervical cancer siHa cell apoptosis by a flow cytometry assay, the lipophilic 6″-O-sorboyl-polydatin exhibited improved apoptosis-inducing capability than the parent drug. The presence of the more lipophilic sorboyl chain in the acylated derivative could account for this.

Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb.

Coumarin-appended phosphorescent cyclometalated iridium(iii) complexes as mitochondria-targeted theranostic anticancer agents.

Theranostic anticancer agents incorporating anticancer properties with capabilities for real-time treatment assessment are appealing candidates for chemotherapy. The design of mitochondria-targeted cytotoxic drugs represents a promising approach to target tumors selectively and overcome resistance to current anticancer therapies. In this work, three coumarin-appended phosphorescent cyclometalated iridium(iii) complexes 1-3 have been explored as mitochondria-targeted theranostic anticancer agents. These complexes display rich photophysical properties, which facilitate the study of their intracellular fate. All three complexes can specifically target mitochondria and show much higher antiproliferative activities than cisplatin against various cancer cells including cisplatin-resistant cells. 1-3 can penetrate into human cervical carcinoma (HeLa) cells quickly and efficiently, and they can carry out theranostic functions by simultaneously inducing and monitoring the morphological changes in mitochondria. Mechanism studies show that 1-3 exert their anticancer efficacy by initiating a cascade of events related to mitochondrial dysfunction. Genome-wide transcriptional and Connectivity Map analyses reveal that the cytotoxicity of complex 3 is associated with pathways involved in mitochondrial dysfunction and apoptosis.

Chemopreventive and Anticancer Activities of Allium victorialis var. platyphyllum Extracts.

Background:Allium victorialis var. platyphyllum is an edible perennial herb and has been used as a vegetable or as a Korean traditional medicine. Allium species have received much attention owing to their diverse pharmacological properties, including antioxidative, anti-inflammatory, and anticancer activities. However, A. victorialis var. platyphyllum needs more study.Methods:The chemopreventive potential of A. victorialis var. platyphyllum methanol extracts was examined by measuring 12-O-tetra-decanoylphorbol 13-acetate (TPA)-induced superoxide anion production in the differentiated HL-60 cells, TPA-induced mouse ear edema, and Ames/Salmonella mutagenicity. The apoptosis-inducing capabilities of the extracts were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, 4’,6-diamidino-2-phenylindole staining, and the DNA fragmentation assay in human colon cancer HT-29 cells. Antimetastatic activities of the extracts were also investigated in an experimental mouse lung metastasis model.Results:The methanol extracts of A. victorialis var. platyphyllum rhizome (AVP-R) and A. victorialis var. platyphyllum stem (AVP-S) dose-dependently inhibited the TPA-induced generation of superoxide anion in HL-60 cells and TPA-induced ear edema in mice, as well as 7,12-dimethylbenz[a]anthracene (DMBA) and tert-butyl hydroperoxide (t-BOOH) -induced bacterial mutagenesis. AVP-R and AVP-S reduced cell viability in a dose-related manner and induced apoptotic morphological changes and internucleosomal DNA fragmentation in HT-29 cells. In the experimental mouse lung metastasis model, the formation of tumor nodules in lung tissue was significantly inhibited by the treatment of the extracts.Conclusions:AVP-R and AVP-S possess antioxidative, anti-inflammatory, antimutagenic, proapoptotic, and antimetastatic activities. Therefore, these extracts can serve as a beneficial supplement for the prevention and treatment of cancer.

Variability in functional p53 reactivation by PRIMA-1Met/APR-246 in Ewing sarcoma

Background:Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients.Methods:PRIMA-1Met, also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations.Results:APR-246 variably induced apoptosis, associated with Noxa, Puma or p21WAF1 upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines.Conclusion:This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.

Rough‐Form Lipopolysaccharide Increases Apoptosis in Human CD4+ and CD8+ T Lymphocytes

Immunosuppression induced by lymphocyte apoptosis is considered an important factor in the pathogenesis of sepsis and has been demonstrated in both animal models of lipopolysaccharide (LPS)-induced endotoxemia and septic patients. As rough-form LPS (R-LPS) has recently been shown to elicit a stronger immunological response than regular smooth-form LPS (S-LPS), we aimed to assess the apoptosis-inducing capabilities of R-LPS in different subsets of lymphocytes (CD4(+) T cells, CD8(+) T cell, B cells and NK cells). Using multicolour flow cytometry on human peripheral blood mononuclear cells, we found that R-LPS increased apoptosis in CD4(+) and CD8(+) T cells assessed by annexin V and propidium iodide (AV(+) PI(-)), compared with both S-LPS-stimulated and unstimulated cells. 7-Amino-actinomycin D and staining for intracellular active caspase-3, which are considered later signs of apoptosis, did not reveal the same results. Both forms appeared to inhibit apoptosis in B cells, but no LPS-form-specific effect was seen on B or NK cells. Our results indicate that R-LPS induces a stronger AV(+) PI(-)-assessed apoptotic response in T cells than S-LPS. Our findings emphasize the importance of T cell apoptosis in endotoxemia and advocates for control of LPS form in both endotoxemia research and clinical trials with Gram-negative infections.

Pleurospermum kamtschaticumExtract Induces ApoptosisviaMitochondrial Pathway and NAG-1 Expression in Colon Cancer Cells

To evaluate the anticarcinogenic activity of methanol extract of Pleurospermum kamtschaticum (PKE), we assessed its apoptosis-inducing capability in HT-29 colon carcinoma cells. PKE treatment for 2 h reduced cell viability in a dose-related manner, and induced apoptotic morphological changes. Flow cytometric analysis indicated that PKE treatment at 0.05 mg/ml induced early apoptosis in 66.2% of HT-29 cells. Additionally, Bcl-2 expression was substantially reduced in PKE-treated HT-29 cells, increasing the Bax/Bcl-2 ratio. The protein levels of procaspase-9 and procaspase-3 were decreased markedly, reflecting caspase-9 and caspase-3 activation, and resulting PARP cleavage was noted in the PKE-treated HT-29 cells. Furthermore, we detected increased NAG-1 expression in the PKE-treated HT-29 cells. In an in vivo study, intraperitoneal PKE administration suppressed the formation of tumor nodules in the lungs of mice. These results indicate that PKE can serve as a beneficial supplement in the treatment and the prevention of colon cancer.

Abstract 5467: Breflate upregulates TRAIL gene transcription in Bax-null cells and induces TRAIL-mediated apoptosis independently of p53 in TRAIL-sensitive tumor lines

Abstract TNF-related apoptosis-inducing ligand (TRAIL) has been hailed as a promising cancer drug target due to its apoptosis-inducing capabilities which are aimed selectively at cancer cells. Methods of inducing endogenous TRAIL as well as introducing exogenous recombinant human TRAIL (rhTRAIL) are being explored in preclinical studies and in the case of the latter, clinical trials. While the gene encoding for DR5, a TRAIL death receptor, has been known to be regulated by p53, we have recently shown that TRAIL is regulated by p53 at base pairs 504-1102 of its promoter (Kuribayashi et al., Cancer Biology and Therapy. 7:2034-2038, 2008). p53 is mutated in over half of all cancers; therefore, a method of upregulating endogenous TRAIL via a mechanism independent of p53 holds great potential therapeutic value for cancer. We searched for small molecule p53-independent upregulators of TRAIL using a luciferase reporter assay which utilized a plasmid with the gene encoding for firefly luciferase under the transcriptional control of the first 504 base pairs of the human TRAIL promoter. Screening with the NCI Diversity Set II library yielded several hits with >2-fold TRAIL induction which were repeated and further analyzed for use as a drug and to gain mechanistic insight. Interestingly, one hit from this screen was breflate, the prodrug of Brefeldin A (BFA). BFA inhibits retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum and is currently being investigated as a cancer therapeutic. Shao et al. (Exp. Cell Res. 227:190-196, 1996) have previously reported that BFA is a potent inducer of apoptosis in some cancer cell lines independently of p53, though the mechanistic relationship was not clear. Breflate clearly increased intracellular levels of TRAIL in HCT116 Bax−/− cells as determined by Western blot analysis, and in addition to the original reporter assay this is being corroborated by reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, sub-G1 analysis found that breflate induced levels of apoptosis >2-fold relative to control levels at 10μM after 24 hours in HCT116 p53−/− cells. Further mechanistic and ongoing in vivo studies are being conducted with breflate alone and in combination with chemotherapy or targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5467.

Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.

The Apoptosis-Inducing Effects of HIV vpr Recombinant Eukaryotic Expression Vectors with Different Mutation Sites on Transfected Hela Cells

The vpr gene of human immunodeficiency virus type 1 (HIV-1) plays an important role in the pathogenicity of viruses. Previous studies showed that mutation of certain sites in HIV-1 Vpr amino acid sequences might influence the clinical process of infected individuals and attenuate its apoptosis-inducing capacity on infected cells. The present study was designed to transfect hela cells with several HIV-1 vpr fragments carrying specific mutation sites, and to observe the distinction of apoptosis-inducing effects of different HIV-1 vpr variant fragments on host cells and explore the possible mechanisms. According to the previous results, 14 typical vpr variant fragments were chosen from Chinese HIV-1 infected individuals. After PCR amplification of vpr gene, the products were purified and double digested with Hind and BamHI. Then pcDNA3.1 (+) eukaryotic expression plasmids were used for the ligation and transduction experiments. The recombinant plasmids were transiently transfected into Hela cells with liposomes, and meanwhile the blank cell and empty vector cell were established as control. RT-PCR was used to detect the mRNA expression of target genes; fluorescent microscope for observing apoptotic cells with Hoechst staining; DNA agarose electrophoresis for detecting apoptotic gradient band; Annexin assay for detecting cell apoptosis; and Caspase activity test for exploring the pathway of cell apoptosis. After transfected with 14 vpr gene segments, the Hela cells exhibited various apoptosis-inducing capabilities. We found that the HIV-1 Vpr segments with mutation at the 70(th), 85(th), 86(th), or 94(th) site showed lower apoptosis-inducing capabilities than other segments on Hela cells, and the apoptosis-inducing ability of HIV-1 vpr gene on host cells might be related to its subtype. Meanwhile, we found that the Caspase3 activity of transfected cell carrying these mutated fragment sites was decreased as compared to the cell with other fragments. This study firstly found that the HIV-1 Vpr fragments with the 70(th), 85(th), 86(th), or 94(th) site mutation might attenuate their apoptosis-inducing abilities on Hela cells. One of the mechanisms might be the attenuation of Caspase-3 activity.