Mechanism of Triptolide‐induced Apoptosis: Effect on Caspase Activation and Bid Cleavage and Essentiality of the Hydroxyl Group of Triptolide

Triptolide is a compound extracted from the Chinese herb Tripterygium wilfordii Hook. f. Triptolide has potent anticancer activity. However, the mechanisms by which triptolide exerts its anticancer activities remain unclear. To explore the molecular mechanisms involved in the anticancer activity of triptolide, we have examined the effect of triptolide on the growth of pancreatic carcinoma PANC-1 and cervical adenocarcinoma HeLa cells. We found that treatment of both HeLa and PANC-1 cells with triptolide potently suppressed cell growth and induced apoptosis, indicated by nuclear fragmentation and blebbing. In both HeLa and PANC-1 cells, apoptosis induced by triptolide was associated with activation of both caspase-3 and caspase-8, and cleavage of poly(ADP-ribose) polymerase and Bid. Moreover, in HeLa cells, caspase-9 is also significantly activated in response to triptolide. Overexpression of Bcl-2 in HeLa cells substantially attenuated triptolide-induced apoptosis. Interestingly, substitution of the 14-OH of triptolide with an acetyl group abrogated both its anticancer and its antiinflammatory activities. Our studies suggest that triptolide may exert its anticancer effects by initiating apoptosis through both death-receptor- and mitochondria-mediated pathways. Our results indicate that both the apoptosis-promoting and the antiinflammatory activities of triptolide depend on the 14-OH group.

HMGA2 Maintains Oncogenic RAS-Induced Epithelial-Mesenchymal Transition in Human Pancreatic Cancer Cells

Synthesis and evaluation of curcumin-related compounds for anticancer activity

Antineoplastic activity of a novel ruthenium complex against human hepatocellular carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) cells

Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Piperine (1-piperoylpeperdine), a nitrogenous pungent substance, is present in the fruits of black pepper (Piper nigrum Linn.) and long pepper (Piper longum Linn.). It possesses several pharmacological properties and has been extensively explored for its anti-cancerous activities. The mechanism underlying its anti-cancer potential in human cervical adenocarcinoma (HeLa) cells is not well interpreted. The anti-proliferative effect and the mode of action of piperine were investigated through some potent markers of apoptosis viz.reactive oxygen species (ROS) generation, cellular apoptosis and loss of mitochondrial membrane potential (MMP). DNA fragmentation, cell cycle kinetics, caspase-3 activity and cell migration assays were also conducted to observe the efficacy of piperine against HeLa cells. The results showed that piperine exposure induces apoptosis significantly in a dose-dependent manner and inhibits the growth of HeLa cells with an increase in ROS generation, nuclear condensation and delayed wound healing. In addition, piperine also encourages cell death by the loss of MMP, DNA fragmentation and the activation of caspase-3. Growth inhibition of HeLa cells was found to be associated with G2/M phase arrest and sub-G1 accumulation. The present study provides useful insight into the apoptotic potential of piperine and further in vivo and clinical studies will be needed for its validation and in the finding of more effective and least toxic regimens against cervical cancer.

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. We have found that the collagen microenvironment promotes expression of histone acetyltransferases (HATs) and thereby promotes chemo-resistance; however, HATs have proven to be difficult ‘druggable’ targets. Bromodomain proteins are chromatin readers that can bind to acetylated histones and thereby regulate gene expression. JQ1, an inhibitor of BET class of human bromodomains, was shown to have efficacy against leukemia and lymphoma cell lines. Thus, we examined the effect of JQ1 on a panel of PDAC cells grown in 3D collagen. AsPC1, CD18 and Panc02.03 cells were particularly sensitive to the effects of JQ1, while Panc1 cells were relatively resistant to JQ1 treatment. JQ1 decreased myc mRNA and increased p21 mRNA in CD18 cells, but not in Panc1 cells. JQ1 also decreased growth of chemo-resistant CD18 cells grown in 3D collagen, and blocked EGF and TGF-β1-induced growth and scattering of Panc1 and CD18 cells in 3D collagen. The MEK1/2 inhibitor U0126 and JQ1 synergistically attenuated growth of CD18 and Panc1 cells in 3D collagen. In addition, EGFR inhibitors (AG1478 and PD153035) and JQ1 also synergistically attenuated the growth of CD18 and Panc1 cells in 3D collagen. In contrast, the p38 MAPK inhibitor SB202190 did not attenuate the effect of JQ1 on growth of CD18 and Panc1 cells in 3D collagen. Finally, JQ1 potentiated the effect of gemcitabine on the growth of CD18 and Panc1 cells in 3D collagen. Overall, these results suggest that JQ1 and other bromodomain inhibitors are potential therapeutic agents for the treatment of pancreatic cancer. Citation Format: Vaibhav Sahai, Krishan Kumar, Kazumi Ebine, Halla Nimeiri, Hidayatullah G. Munshi. The bromodomain inhibitor JQ1 blocks growth of pancreatic cancer cells in 3D collagen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2013-2449

The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca2+]er is an important component of the anti-apoptotic program controlled by this oncogene.

BackgroundEichhornia crassipes (Mart) solms is an invasive macrophyte causing serious problems to the network of irrigation and drainage canals in the Nile Delta region. The present study aim to evaluate the potential anticancer and antioxidant activities of Eichhornia crassipes crude extract and its pure compounds.MethodsThe macrophyte was collected from El-Zomor canal, River Nile (Egypt), cleaned, air dried, grinded then extracted with methanol (crude extract). The extract was fractionated using pre-coated silica gel plates (TLC F254) with hexane/ethyl acetate (8.5: 1.5 v/v) as mobile phase. Nine fractions were separated (A-I) then scratched, eluted with the same mobile phase, filtered and the separated fractions were determined and identified using spectroscopic methods (Mass spectrum (MS), Infra red (IR) and Proton H-Nuclear magnetic resonance (H-NMR). Both the crude extract and its nine identified compounds were tested for their antioxidant (using 2, 2 diphenyl-1-picrylhydrazyl (DPPH), 2, 2′- azino-bis {ethylbenzthiazoline-6-sulfonic acid (ABTS.)} methods) and anticancer activity (using MCF-7, HeLa, Hep.G2 and EACC cell lines).ResultsThe antioxidant and anticancer activities of the crude extract exhibited the highest effect while the compounds showed variable effects which depend on the type of compound and cancer cell line. The antioxidant activity of the crude extract exhibited the highest followed in descending order by compounds D, E, G and H respectively. Concerning the anticancer potency, the crude extract showed also the highest effect while the identified compounds (A, B, C, D, E, F, G, H and I) showed variable anticancer activities against the four different cell lines. In addition, Compound I exhibited the most potent anticancer activity against HepG2 cell line while compound D exhibited high anticancer activity against HeLa cells and EACC. The results revealed the presence of different compounds (Alkaloids and terpenoids) with variable antioxidant and anticancer activities which elicited an auto-augmentation in the crude extract leading to its greatest activities. The action of the identified anticancer compounds on DNA fragmentation was studied.ConclusionThe study illustrated the potential of Eichhornia as a valuable resource for natural compounds of desirable medicinal properties (e.g. antioxidants and anticancer).

Anticancer activity in HeLa and MCF-7 cells via apoptopic cell death by a sterol molecule Cholesta-4,6-dien-3-ol (EK-7), from the marine ascidian Eudistoma kaverium

Gemcitabine (Gem) is approved for use in pancreatic cancer chemotherapy. However, Gem undergoes rapid metabolism in the blood, producing an inactive metabolite. Due to this rapid metabolism, the effective dose of Gem is high, thereby predisposing patients to severe adverse effects. This study aimed to improve Gem's metabolic and therapeutic stability by modifying the amine group (4-NH2) with hydroxylamine to form 4-N-hydroxylGem hydrochloride (GemAGY). Micro-elemental analysis and Nuclear Magnetic Resonance (NMR) were used to characterize GemAGY, and its anticancer activity was investigated against MiaPaCa-2, BxPC-3, and PANC-1 pancreatic cancer cell lines. The GemAGY metabolic stability was evaluated in human liver microsomal solution. In the 2D cytotoxicity assay, the IC50 values of GemAGY-treated MiaPaCa-2, PANC-1, and BxPC-3 cells were significantly lower when compared to GemHCl-treated cultures. More so, in 3D spheroid assay results, GemAGY IC50 values were found to be 9.5 ± 1.1 µM and 12.6 ± 1.0 µM when compared to GemHCl IC50 values of 24.1 ± 1.6 µM and 30.2 ± 1.8 µM in MiaPaCa-2 and PANC-1 cells, respectively. GemAGY was stable, with 60% remaining intact after 2 hours of digestion in microsomal enzymes, compared to GemHCl, which had less than 45% remaining intact after 30 minutes. GemAGY-treated MiaPaCa-2 and PANC-1 cells at 3.12 and 6.25 μM concentrations demonstrated a significantly reduced cell migration towards the wound area compared to the GemHCl-treated cultures at the same concentrations. Further, GemAGY-treated MiaPaCa-2 cells significantly increased the expression of p53 and BAX compared to GemHCl-treated cells. GemAGY demonstrated significant anticancer activity and improved metabolic stability compared to GemHCl and is most likely to have potential anticancer activity against pancreatic cancer.

The high mortality of pancreatic ductal adenocarcinoma (PDAC) underscores the need for novel treatments. We investigated the potential role of human biofield therapy (BT) in modifying tumorigenic processes in murine and human PDAC cells through a series of invitro and invivo studies. Cell viability and ultrastructure changes were examined by PrestoBlue assay and Transmission Electron Microscope, respectively. Cell voltage potentials were assessed using DiBAC4 staining. Both FOXM1 CRISPR KO and Tet-on FOXM1 overexpressing PDAC cells were used for the mechanistic studies. Antitumor efficacy was tested in orthotopic models using human PANC-1 and mouse KPCY cells. Multiple mouse and human PDAC cell lines exposed to BT (three different therapists using a standardized procedure) showed significantly reduced proliferation compared to sham control (SC) and incubator control (IC) cells across multiple studies. BT induced swollen mitochondria in PANC-1 cells and increased G1 cell cycle arrest in PANC-1, L3.7, and COLO357 cells. BT also downregulated key cell cycle regulators including FOXM1 in PANC-1 and L3.7 cells (p < 0.05). BT led to a 36.7% reduction of cell voltage potential measured in PANC-1 cells (p < 0.01), with similar effects in MiaPaCa-2 and KPCY cells. Furthermore, BT reduced the invasiveness in PANC-1, L3.7, and COLO357 cells, an effect influenced by FOXM1 gene expression. BT inhibited the primary tumor growth and liver metastasis of PANC-1 and KPCY cells in orthotopic mouse models (p < 0.05). These novel findings support the potential of BT to influencegrowth and metastasis of pancreatic cancer cells and warrant further investigation.