Correction: Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation.

[This corrects the article DOI: 10.1371/journal.pone.0009996.].

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production.

AIMS: We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. RESULTS: HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. INNOVATION AND CONCLUSION: These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation.

BACKGROUND: Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions. METHODOLOGY/PRINCIPAL FINDINGS: We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H(2)O(2)) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H(2)O(2)-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H(2)O(2). Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells. CONCLUSIONS/SIGNIFICANCE: Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.

Dominant negative Rac1 attenuates paclitaxel-induced apoptosis in human melanoma cells through upregulation of heat shock protein 27: a functional proteomic analysis.

GTPase ras-related C3 botulinum toxin substrate 1 (Rac1) plays a role in various cellular processes pertinent to cancer development. In the present study, we investigated the molecular mechanisms underlying apoptosis regulation by Rac1 through functional proteomic analysis of three human melanoma M14 cell lines stably transfected with constitutively active Rac1V12, dominant negative Rac1N17, and empty vector (pIRES), respectively. We found that paclitaxel evoked apoptosis in the melanoma cell lines through the intrinsic (mitochondria) pathway in a caspsae-3-dependent manner. Compared to the Rac1pIRES and Rac1V12 cells, Rac1N17 cells were more resistant to paclitaxel-triggered caspase-3 activation and apoptosis. Protein composition comparisons amongst the three cell lines identified two peptide spots of interest. One was Hsp27, which was upregulated in Rac1N17 cells as assessed in our gel image interpretation, PMF and Western blot analysis. The other was identified as SR-25 protein (also known as the ADP-ribosylation factor-like factor 6-interacting protein 4; ARL6IP4) using PMF, which was separated only from the Rac1N17 cells under the experimental conditions. Moreover, knockdown of the protein level of Hsp27 using small interfering RNA in Rac1N17 cells significantly increased the paclitaxel-elicited caspase-3 activation and apoptosis. In conclusion, our results implicate that Hsp27 and SR-25 are mediators in Rac1 signaling pathway(s). It appears that the dominant negative Rac1N17 reduces the apoptosis sensitivity toward paclitaxel in the melanoma cells through upregulation of Hsp27, which inhibits its down stream drug-elicited caspase-3 activation.

Automated laser scanning cytometry: a powerful tool for multi-parameter analysis of drug-induced apoptosis.

BACKGROUND: Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi-parameter analysis of drug-induced tumor cell apoptosis. MATERIALS: Using 2-mercaptopyridine-N-oxide-hydrate sodium salt, or the commonly used chemotherapeutic agents etoposide and camptothecin, we performed simultaneous analyses of apoptosis-related morphological features as well as fluorescence-based biochemical changes in a 96-well format. RESULTS: We demonstrate the scope of LSC as a platform for comparing multiple variables between different cell populations, distinguishing unique events at a single cell level within a sample population, and enabling simultaneous screenings in a single assay at multiple dosages and time-points. CONCLUSION: These data underscore the power of LSC for simultaneous multi-parameter analysis, which could have implications for screening or assessing the efficacy of drug responses in heterogeneous cell populations and at the single cell level.

Evidence for Oldenlandia diffusa-evoked cancer cell apoptosis through superoxide burst and caspase activation.

BACKGROUND & OBJECTIVE: Oldenlandia diffusa (Bai Hua She She Cao) is one of the herbs most commonly used in traditional Chinese medicine for treating cancer. Various studies using the herb alone or in combination with other therapy plans have evidenced the effectiveness of the herb in the management of cancers of different tissue origin. However, the mechanisms underlying its anti-cancer activity are unknown. In the present study, we attempted to investigate the apoptotic activity of crude extracts of the herb as well as the possible molecular pathways. METHODS: We incubated human promyelocytic leukemia cell line HL60 cells with ethanol or aqueous extracts of the herb, and determined the levels of intracellular superoxide at 2 and 4 hours as well as caspase activity at 3, 6 and 8 hours using photospectrometry. Cancer cell survival and apoptosis were quantified at 24 hours by using MTT and flow cytometry analyses respectively. RESULTS: We found that it dose-dependently inhibited the cancer cell growth in MTT assay. Flow cytometry analysis revealed that it elicited significant production of sub-G(1) population of the cells, indicating the extract-evoked cell apoptotic death. The LD(50) of the ethanol extract was estimated to be approximately 320 microg/ml. Moreover, treatment of the cancer cells with the ethanol component markedly increased the production of superoxide within few hours. Significant elevation in the protease activities of caspases-2 and -3 were detected at as early as 3 and 6 hours respectively. CONCLUSION: Our results show that the ethanol extract of the herb effectively evokes cancer cell apoptosis, possibly through burst-mediated caspase activation.

Evidence for Oldenlandia diffusa-evoked cancer cell apoptosis through superoxide burst and caspase activation / 中西医结合学报

BACKGROUND & OBJECTIVE: Oldenlandia diffusa (Bai Hua She She Cao) is one of the herbs most commonly used in traditional Chinese medicine for treating cancer. Various studies using the herb alone or in combination with other therapy plans have evidenced the effectiveness of the herb in the management of cancers of different tissue origin. However, the mechanisms underlying its anti-cancer activity are unknown. In the present study, we attempted to investigate the apoptotic activity of crude extracts of the herb as well as the possible molecular pathways. METHODS: We incubated human promyelocytic leukemia cell line HL60 cells with ethanol or aqueous extracts of the herb, and determined the levels of intracellular superoxide at 2 and 4 hours as well as caspase activity at 3, 6 and 8 hours using photospectrometry. Cancer cell survival and apoptosis were quantified at 24 hours by using MTT and flow cytometry analyses respectively. RESULTS: We found that it dose-dependently inhibited the cancer cell growth in MTT assay. Flow cytometry analysis revealed that it elicited significant production of sub-G(1) population of the cells, indicating the extract-evoked cell apoptotic death. The LD(50) of the ethanol extract was estimated to be approximately 320 microg/ml. Moreover, treatment of the cancer cells with the ethanol component markedly increased the production of superoxide within few hours. Significant elevation in the protease activities of caspases-2 and -3 were detected at as early as 3 and 6 hours respectively. CONCLUSION: Our results show that the ethanol extract of the herb effectively evokes cancer cell apoptosis, possibly through burst-mediated caspase activation.