bafilomycin-a1 and Colorectal-Neoplasms

Autophagy protects colorectal cancer cells against therapeutic intervention. Autophagy is a continuous process, and autophagic flux requires both autophagosome synthesis and their subsequent degradation at lysosomes. Hence, cells with elevated autophagic flux display both rapid autophagosome generation and degradation. Here, we describe an immunoblot protocol coupled to pharmaceutical inhibition of autophagosome clearance to monitor autophagic flux levels between colorectal cancer cell lines.

Topics: Autophagy; Blotting, Western; Cell Culture Techniques; Cell Line, Tumor; Colorectal Neoplasms; Humans; Lipopeptides; Lysosomes; Macrolides; Microtubule-Associated Proteins; Phagosomes

Previous studies by our group have demonstrated that extract of clove exhibits potent anticancer effects in vitro and in vivo. In the present study, the effect of an extracted and isolated active fraction of clove (AFC) on induction of cellular apoptosis in human colorectal cancer HCT-116 cells was investigated by morphological observation, flow cytometry, and western blotting analysis. The results revealed that AFC induced apoptosis of HCT-116 cells. AFC also induced autophagy, demonstrated by increased punctuate microtubule-associated protein 1A/1B-light chain 3 (LC3) staining, and LC3-II and Beclin-1 protein expression levels. Furthermore, the autophagy inhibitors 3-MA and baflomycin A1 potentiated the pro-apoptotic activity of AFC in HCT-116 cells. AFC also inhibited the phosphorylation of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signaling pathway. The present study may improve the existing understanding of the anticancer mechanisms of clove and provide a scientific rationale for AFC to be further developed as a promising novel anticancer agent for the treatment of colorectal cancer.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Macrolides; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Syzygium; TOR Serine-Threonine Kinases

The IDF-11774, a novel clinical candidate for cancer therapy, targets HSP70 and inhibits mitochondrial respiration, resulting in the activation of AMPK and reduction in HIF-1α accumulation.. To identify genes that have synthetic lethality to IDF-11774, RNA interference screening was conducted, using pooled lentiviruses expressing a short hairpin RNA library.. We identified ATP6V0C, encoding the V0 subunit C of lysosomal V-ATPase, knockdown of which induced a synergistic growth-inhibitory effect in HCT116 cells in the presence of IDF-11774. The synthetic lethality of IDF-11774 with ATP6V0C possibly correlates with IDF-11774-mediated autolysosome formation. Notably, the synergistic effect of IDF-11774 and the ATP6V0C inhibitor, bafilomycin A1, depended on the PIK3CA genetic status and Bcl-2 expression, which regulates autolysosome formation and apoptosis. Similarly, in an experiment using conditionally reprogramed cells derived from colorectal cancer patients, synergistic growth inhibition was observed in cells with low Bcl-2 expression.. Bcl-2 is a biomarker for the synthetic lethal interaction of IDF-11774 with ATP6V0C, which is clinically applicable for the treatment of cancer patients with IDF-11774 or autophagy-inducing anti-cancer drugs.

Topics: Adamantane; Animals; Apoptosis; Autophagy; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrolides; Mice; Piperazines; Proto-Oncogene Proteins c-bcl-2; Vacuolar Proton-Translocating ATPases; Xenograft Model Antitumor Assays

Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Beclin-1; Caco-2 Cells; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; HT29 Cells; Humans; Indoles; Macrolides; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Sequestosome-1 Protein; Sulfonamides; TOR Serine-Threonine Kinases

Small molecules with the potential to initiate different types of programmed cell death could be useful 'adjunct therapy' where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨm) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT-MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly 'double-edged sword' against CRC cells thereby further advocating its clinical effectiveness against this malignancy.

Topics: Adenine; AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Shape; Cell Survival; Colorectal Neoplasms; Endoplasmic Reticulum Stress; Energy Metabolism; Enzyme Activation; Epidermal Growth Factor; Humans; Macrolides; Metabolomics; Mice; Mitogens; Models, Biological; Protein Biosynthesis; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Silybin; Silymarin; Somatomedins; TOR Serine-Threonine Kinases; Vacuoles