rottlerin and Lung-Neoplasms

Rottlerin, a polyphenolic compound derived from Mallotus philipinensis, has been reported to exhibit anti-tumor activities in a variety of human malignancies including NSCLC (non-small cell lung cancer). TAZ (transcriptional co-activator with PDZ-binding motif), one of the key activators in Hippo pathway, has been characterized as an oncoprotein. Therefore, inhibition of TAZ could be useful for the treatment of human cancers. In the current study, we aimed to explore whether rottlerin inhibits the expression of TAZ in NSCLC, leading to its anti-cancer activity. Multiple approaches were applied for determining the mechanism of rottlerin-mediated anti-tumor function, including cell growth assay, Flow cytometry, wound healing assay, invasion assay, Western blotting, and transfection. We found that rottlerin inhibited cell growth, triggered apoptosis, arrested cell cycle, and retarded cell invasion in NSCLC cells. Moreover, our results showed that overexpression of TAZ enhanced cell growth, stimulated apoptosis, and promoted cell migration and invasion. Consistently, inhibition of TAZ exhibited anti-tumor activity in NSCLC cells. Notably, we validated that rottlerin exerted its tumor suppressive function via inactivation of TAZ in NSCLC cells. Taken together, our study indicates that inhibition of TAZ by rottlerin could be a promising strategy for the prevention and therapy of NSCLC.

Topics: A549 Cells; Acetophenones; Antineoplastic Agents, Phytogenic; Benzopyrans; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Neoplasm Invasiveness; Signal Transduction; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transfection

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone-independent mammary cancer cell line LM38-LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38-LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic-vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self-renewal. Furthermore, Rottlerin pre-treatment induced in CSC the development of a "grape-like" morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self-renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self-renewal.

Topics: Acetophenones; Animals; Antineoplastic Agents; Autophagy; Benzopyrans; Cell Line, Tumor; Cell Proliferation; Cell Self Renewal; Drug Screening Assays, Antitumor; Female; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Nanog Homeobox Protein; Neoplasm Transplantation; Neoplastic Stem Cells; Protein Kinase C-delta; Protein Kinase Inhibitors

Photodynamic therapy is becoming a widely accepted form of cancer treatment using a photosensitizing agent and light. Our previous study has demonstrated that photoactivated aloe-emodin induced anoikis and changes in cell morphology, which were in part mediated through its effect on cytoskeleton in lung carcinoma H460 cells. However, the molecular mechanisms of these photoactivated aloe-emodin-induced changes remain unknown. The present study demonstrated that the expression of protein kinase Cδ (PKCδ) was triggered by aloe-emodin and irradiation in H460 cells. Furthermore, the photoactivated aloe-emodin-induced cell death and translocation of PKCδ from the cytosol to the nucleus was found to be significantly inhibited by rottlerin, a PKCδ-selective inhibitor. Western blot analysis demonstrated that rottlerin also reversed the decrease in protein expression of cytoskeleton-related proteins, such as rat sarcoma (RAS), ras homolog gene family member A (RHO), p38, heat shock protein 27 (HSP27), focal adhesion kinase (FAK), α-actinin and tubulin, induced by photoactivated aloe-emodin. Our findings suggest that the regulation of cytoskeleton-related proteins mediated by PKCδ may be the mechanisms for the protective effects of rottlerin against the photoactivated aloe-emodin induced H460 cell death.

Topics: Acetophenones; Actin Cytoskeleton; Actins; Anthraquinones; Apoptosis; Benzopyrans; Cell Death; Cell Line, Tumor; Cytoskeleton; Humans; Lung Neoplasms; Photochemotherapy; Protein Kinase C-delta

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K(i)=2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k(inact)) of (0.73 ± 0.07) × 10⁻³ μM⁻¹ min⁻¹. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.

Topics: Acetophenones; Benzopyrans; Binding, Competitive; Enzyme Inhibitors; Escherichia coli; Fluorescence; Hemin; High-Throughput Screening Assays; Humans; Kinetics; Lung Neoplasms; Molecular Targeted Therapy; NADP; Oxidation-Reduction; Protoporphyrins; Recombinant Proteins; Small Molecule Libraries; Sodium Selenite; Thioredoxin Reductase 1; Tumor Cells, Cultured

The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21(Ras)-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI(3)-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21(Ras) signaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24  h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.

Topics: Acetophenones; Animals; Apoptosis; Benzopyrans; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Gastrointestinal Neoplasms; Gene Knockdown Techniques; Humans; Lung Neoplasms; Mice; Neuroendocrine Tumors; NIH 3T3 Cells; Protein Kinase C-delta

Overexpression of the anti-apoptotic protein Bcl-2 has been associated with several malignancies, including small cell lung cancer (SCLC). In the present study, we have investigated if Bcl-2 contributes to the emergence of cisplatin resistance in SCLC H69 cells. The ability of cisplatin to induce apoptosis was decreased in H69 cells that acquired resistance to cisplatin (H69/CP). The level of Bcl-2 was, however, substantially reduced in H69/CP cells compared to parental H69 cells. There was little change in Bcl-2 content in H69 cells that were resistant to etoposide (VP-16) or Taxol. Bcl-2 was constitutively phosphorylated at serine 70 in H69 cells but not in H69/CP cells and cisplatin had little effect on Bcl-2 phosphorylation. The level of procaspase-3 was elevated in H69/CP cells but the ability of cisplatin to induce mitochondrial depolarization, caspase-9 activation, and poly(ADP-ribose) polymerase (PARP) cleavage was compromised in H69/CP cells. The level of the anti-apoptotic protein Bcl-x(L) and the pro-apoptotic protein Bax was slightly reduced in H69/CP cells but the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins was not sufficient to explain cellular resistance to cisplatin. These results suggest that the acquisition of cisplatin resistance by H69 cells was not due to an increase in the level/phosphorylation status of the anti-apoptotic protein Bcl-2.

Topics: Acetophenones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzopyrans; Blotting, Western; Carcinoma, Small Cell; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Etoposide; Flow Cytometry; Humans; Immunoblotting; Lung Neoplasms; Membrane Potentials; Mitochondria; Paclitaxel; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

Drugs that target protein kinase C (PKC) are now being evaluated in patients with non-small cell lung cancer (NSCLC), but the role of PKC in NSCLC cells remains unclear. We report here that NSCLC cell lines show enhanced phosphorylation and altered expression of specific PKC isoforms compared with normal lung epithelial cells. PKC inhibition variably increased apoptosis, with rottlerin, a PKCdelta inhibitor, being most effective and potentiating chemotherapy-induced apoptosis, especially with trastuzumab. Consistent with PKCdelta being anti-apoptotic in NSCLC cells, transient transfection of a kinase-dead mutant of PKCdelta increased apoptosis and potentiated chemotherapy-induced apoptosis. Our studies provide a rationale for targeting PKC isoforms in NSCLC cells, especially PKCdelta.

Topics: Acetophenones; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzopyrans; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Humans; Isoenzymes; Lung Neoplasms; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Transfection; Trastuzumab