Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage

Background and PurposeAshwagandha is a popular Ayurvedic herb used in Indian traditional home medicine. It has been assigned a variety of health-promoting effects of which the mechanisms remain unknown. We previously reported the selective killing of cancer cells by leaf extract of Ashwagandha (i-Extract) and its purified component Withanone. In the present study, we investigated its mechanism by loss-of-function screening (abrogation of i-Extract induced cancer cell killing) of the cellular targets and gene pathways.Methodology/Principal FindingsRandomized ribozyme library was introduced into cancer cells prior to the treatment with i-Extract. Ribozymes were recovered from cells that survived the i-Extract treatment. Gene targets of the selected ribozymes (as predicted by database search) were analyzed by bioinformatics and pathway analyses. The targets were validated for their role in i-Extract induced selective killing of cancer cells by biochemical and molecular assays. Fifteen gene-targets were identified and were investigated for their role in specific cancer cell killing activity of i-Extract and its two major components (Withaferin A and Withanone) by undertaking the shRNA-mediated gene silencing approach. Bioinformatics on the selected gene-targets revealed the involvement of p53, apoptosis and insulin/IGF signaling pathways linked to the ROS signaling. We examined the involvement of ROS-signaling components (ROS levels, DNA damage, mitochondrial structure and membrane potential) and demonstrate that the selective killing of cancer cells is mediated by induction of oxidative stress.ConclusionAshwagandha leaf extract and Withanone cause selective killing of cancer cells by induction of ROS-signaling and hence are potential reagents that could be recruited for ROS-mediated cancer chemotherapy.

Effective targeting by uniformly functionalized nanoparticles is limited to cancer cells expressing at least two copies of targeted receptors per nanoparticle footprint (approximately ≥2 × 10(5) receptor copies per cell); such a receptor density supports the required multivalent interaction between the neighboring receptors and the ligands from a single nanoparticle. To enable selective targeting below this receptor density, ligands on the surface of lipid vesicles were displayed in clusters that were designed to form at the acidic pH of the tumor interstitium. Vesicles with clustered HER2-targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized vesicles. On HER2-negative breast cancer cells MDA-MB-231 and MCF7 {expressing (8.3 ± 0.8) × 10(4) and (5.4 ± 0.9) × 10(4) HER2 copies per cell, respectively}, only the sticky vesicles exhibited detectable specific targeting (KD ≈ 49-69 nM); dissociation (0.005-0.009 min(-1)) and endocytosis rates (0.024-0.026 min(-1)) were independent of HER2 expression for these cells. MDA-MB-231 and MCF7 were killed only by sticky vesicles encapsulating doxorubicin (32-40% viability) or α-particle emitter (225)Ac (39-58% viability) and were not affected by uniformly functionalized vesicles (>80% viability). Toxicities on cardiomyocytes and normal breast cells (expressing HER2 at considerably lower but not insignificant levels) were not observed, suggesting the potential of tunable clustered ligand display for the selective killing of cancer cells with low receptor densities.

Mitochondrial dysfunction is an important mechanism by which cancer cells become resistant to chemotherapeutic agents. Oncogenic Ras activation is often observed in human cancer and was reported to cause mitochondrial dysfunction by compromising mitochondrial electron transport chain. Therefore, identification of new agents capable of effectively killing mitochondrial defective cancer cells would have important therapeutic implications. Our previous work has discovered that mitochondrial respiratory dysfunction could cause increased NADPH oxidase (NOX) activity to support cell survival in the metabolic switch from mitochondrial oxidative phosphorylation to glycolysis. Mitochondrial respiratory dysfunction can also cause profound redox alteration to adapt ROS stress. Here, we report that targeted inhibition of NOX and glutathione synthesis has synergy effect in killing mitochondrial respiratory dysfunction cells, including cancer cells harboring oncogenic Ras activation. Activation of NOX and increased glutathione antioxidants are always observed in Ras activated cells. Treatment of these cancer cells with DPI and BSO to inhibit NOX activity and antioxidant adaptation function resulted in an elevated ROS stress, increased gamma-H2AX expression, leading to massive death of the cancer cells, such as Panc-1 and HCT116 cells. Antioxidant NAC and catalase attenuated the BSO and DPI induced cell death in mitochondrial respiratory dysfunction cells. The synergistic effect of BSO and DPI to kill oncogenic Ras bearing HCT116 cells was further confirmed using colony formation assay. Our results suggest that DPI, in synergy with BSO, is a promising compound capable of killing cancer cells bearing oncogenic Ras mutations through a ROS-mediated mechanism and warrants further investigations. Citation Format: Weiqin Lu, Yaying Yang, Craig Logsdon, Peng Huang. Selective killing of cancer cells with mitochondrial respiratory dysfunction induced by activation of oncogenic Ras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2014-806

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells.

Conventional genotoxic anti-cancer drugs target the proliferative advantage of tumor cells over normal cells. This kind of approach lacks the selectivity of treatment to cancer cells, because most of the targeted pathways are essential for the survival of normal cells. As a result, traditional cancer treatments are often limited by undesirable damage to normal cells (side-effects). Ideal anti-cancer drugs are expected to be highly effective against malignant tumor cells with minimal cytotoxicity toward normal cells. Such selective killing can be achieved by targeting pathways essential for the survival of cancer cells, but not normal cells. As cancer cells are characterized by their resistance to apoptosis, selective apoptosis induction is a promising approach for selective killing of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising tumor-selective anti-cancer drug. However, the congenital and acquired resistance of some cancer cell types, including malignant melanoma cells, currently impedes effective TRAIL therapy, and an innovative approach that can override TRAIL resistance is urgently required. Apoptosis is characterized by cell shrinkage caused by disruption of the maintenance of the normal physiological concentrations of K+ and Na+ and intracellular ion homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis. Recent evidence suggests that depolarization is an early and prerequisite event during TRAIL-induced apoptosis. Moreover, diverse natural products and synthetic chemicals capable of depolarizing the cell membrane exhibit tumor-selective killing and TRAIL-sensitizing effects. Here, we discuss the role of depolarization in selective killing of cancer cells in connection with the emerging concept that oxidative stress is a critical mediator of mitochondrial and endoplasmic reticulum dysfunctions and serves as a tumor-selective target in cancer treatment.

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

Mutations of multiple tumor suppressor genes, such as PTEN and p53, have been proposed to play important roles in the development of prostate cancer. Loss of one allele of PTEN occurs in 70-80% of human primary prostate tumors and homozygous inactivation of PTEN is associated with advanced disease. Similarly, p53 is found completely lost or mutated almost exclusively in advanced human prostate cancer. Thus, selective killing of prostate cancer cells harboring mutations of PTEN and p53 may prove to be a promising strategy for the treatment of advanced prostate cancer. The Warburg effect of aerobic glycolysis has now been generally accepted as a key metabolic hallmark of cancer. In this study, we investigated the molecular target leading to the Warburg effect in the growth and aggressiveness of prostate cancer cells harboring inactivation of PTEN and p53 and delineated the underlying mechanism. We identified that expression of hexokinase II (HK2), an enzyme involved in the first step of glycolysis, is preferentially elevated in human prostate cancer cells bearing mutations of both PTEN and p53 (PC3). Functional studies demonstrated that HK2 expression is crucial for the Warburg effect in PC3 cells and knockdown of HK2 inhibits tumor growth in PC3 xenograft mouse model. These novel findings prompted us to test whether 2-de-oxyglucose (2-DG), an inhibitor of HK2, could potentially suppress prostate cancer growth by targeting the Warburg effect. We found that an induction of AMPK-dependent autophagy prevents cancer cells from apoptosis upon 2-DG treatment, thereby limiting therapeutic efficacy on prostate cancer in vivo. Consistent with cell survival function of autophagy, its inhibition by chloroquine (a small molecule inhibitor of autophagy) or individual knockdown of the essential genes involved in autophagy (Atg5, Atg7, Beclin1, and ULK1) induced massive cell death when combined with 2-DG. This cell death can be rescued by overexpression of anti-apoptotic protein Bcl-2 or downregulation of pro-apoptotic protein Bax in PC3. More importantly, we demonstrated that combination of chloroquine and 2-DG could specifically kill prostate cancer cells, leaving normal prostate epithelial cells untouched. This specificity is due to the preferential induction of HK2 through the activated Rictor-AKT-mTOR pathway in cancer cells. Finally, combination of chloroquine and 2-DG caused synthetic lethality in prostate cancer cells and effectively suppressed tumor growth in PC3 xenograft mouse model. Towards therapeutic translation, we have observed that expression of HK2 by staining primary human prostate tumor samples with HK2 antibodies correlated with the stages of prostate cancer. Given our findings, we therefore propose that targeting the Warburg effect and autophagy pathways may serve as an effective and selective treatment for patients with advanced prostate cancer, in particular those with PTEN and p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3213. doi:1538-7445.AM2012-3213

Photothermal therapy (PTT) systems typically do not possess intrinsic tumor-targeting capability, resulting in indiscriminate thermal damage to both cancer and normal cells. Herein, a low-density lipoprotein (LDL)-based nanosystem (denoted as MTTQ@LDL) is reported for targeted photothermal killing of cancer cells. Such a nanosystem is fabricated by reconstituting the lipophilic core of LDL with an organic photothermal agent MTTQ. The reconstitution process improves the supramolecular photothermal effects of MTTQ assemblies, which contributes to the significantly enhanced photothermal conversion efficiency (41.3%vs. 16.2%). MTTQ@LDL can actively target LDL receptor-overexpressed cancer cells via receptor-mediated endocytosis, enabling the selective killing of cancer cells over normal cells (98%vs. 7%) post-NIR irradiation. Reconstituted LDL can serve as a promising platform for targeted delivery of functional materials, holding great promise in tumor eradication in vivo.

Simple SummaryA review of over six decades of cancer chemotherapies, including recent immunotherapy, reveals partial success in the battle against cancer. One of the main reasons for this slow progress is the failure, by mainstream anticancer treatments, to distinguish between cancer cells and normal cells. For this reason, the aim of our study was to assess if sonodynamic therapy (SDT), a new anticancer approach, can affect cancer cells only, avoiding any harmful effects on normal cells. SDT aims to cure malignant tumors by using a chemical compound (sonosensitizer) triggered by ultrasound exposure. For this purpose, the effects on cancer cells and normal cells, namely HT-29 cells and HDF 106-05 cells, subjected to sonodynamic treatment were investigated. Our results show that, according to different plasma membrane properties of cancer cells and normal cells, a different sonodynamic effect occurs, reaching a remarkable cytotoxic effect on cancer cells only.Sonodynamic Therapy (SDT) is a new anticancer strategy based on ultrasound (US) technique and is derived from photodynamic therapy (PDT); SDT is still, however, far from clinical application. In order to move this therapy forward from bench to bedside, investigations have been focused on treatment selectivity between cancer cells and normal cells. As a result, the effects of the porphyrin activation by SDT on cancer (HT-29) and normal (HDF 106-05) cells were studied in a co-culture evaluating cell cytotoxicity, reactive oxygen species (ROS) production, mitochondrial function and plasma membrane fluidity according to the bilayer sonophore (BLS) theory. While PDT induced similar effects on both HT-29 and HDF 106-05 cells in co-culture, SDT elicited significant cytotoxicity, ROS production and mitochondrial impairment on HT-29 cells only, whereas HDF 106-05 cells were unaffected. Notably, HT-29 and HDF 106-05 showed different cell membrane fluidity during US exposure. In conclusion, our data demonstrate a marked difference between cancer cells and normal cells in co-culture in term of responsiveness to SDT, suggesting that this different behavior can be ascribed to diversity in plasma membrane properties, such as membrane fluidity, according to the BLS theory.

Herein, a photosensitizer (riboflavin) tailored surface functionalized carbon dot (RCD1s) was designed to utilize it in visible light induced targeted cancer therapy. At first, phenylboronic acid appended biotinylated blue emitting carbon dot (CD1s) was synthesized. Riboflavin having "diol" moiety was covalently linked with this CD1s to prepare RCD1s by using complementary boronate-diol linkage. Lewis acid-base interaction facilitated the covalent linkage formation between the surface functionalizing agent of CD1s and riboflavin to develop water-soluble, green emitting RCD1s. Interestingly, this newly synthesized RCD1s has the ability to produce reactive oxygen species (ROS) such as hydroxyl and superoxide radicals under exposure of visible light (wavelength: 460-490 nm). These ROS also can destroy the structure of DNA by oxidative pathway. Thus, under irradiation of visible light (wavelength: 460-490 nm), RCD1s was found to kill HeLa and B16F10 melanoma cells over noncancer cell NIH3T3 by ∼5-fold higher efficacy through ROS induced oxidative DNA damage. The presence of biotin on the surface of the riboflavin tethered carbon dot is essential for the selective killing of cancer cells over normal cells. In the presence of UV light (340-420 nm), RCD1s showed no notable killing of cancer cells as well as normal cells. Besides, RCD1s in the presence of visible light selectively stained HeLa and B16F10 cells over noncancerous cell NIH3T3 by exploiting its fluorescence and cancer cell targeting moiety, biotin. Hence, the newly developed RCD1s can be utilized in theranostic applications including bioimaging and selective killing of cancer cells in the presence of visible light (460-490 nm).

Early work in the 1970s by Linus Pauling, a twice-honored Nobel laureate, led to his proposal of using high-dose vitamin C to treat cancer patients. Over the past several decades, a number of studies in animal models as well as several small-scale clinical studies have provided substantial support of Linus Pauling's early proposal. Production of reactive oxygen species (ROS) via oxidation of vitamin C appears to be a major underlying event, leading to the selective killing of cancer cells. However, it remains unclear how vitamin C selectively kills cancer cells while sparing normal cells and what the molecular targets of high-dose vitamin C are. In a recent article published in Science (2015 December 11; 350(6266):1391-6. doi: 10.1126/science.aaa5004), Yun et al. reported that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) through an ROS-dependent mechanism. This work by Yun et al. along with other findings advances our current understanding of the molecular basis of high-dose vitamin C-mediated cancer cell killing, which will likely give an impetus to the continued research efforts aiming to further decipher the novel biochemistry of vitamin C and its unique role in cancer therapy.

Investigation of the survival viability of cervical cancer cells (HeLa) under visible light induced photo-catalysis with facile synthesized WO3/ZnO nanocomposite☆

Doxorubicin loaded 17β-estradiol based SWNT dispersions for target specific killing of cancer cells

Biomimetic pro-apoptotic agents (e.g., BH3 mimetics) have been shown to activate the intrinsic death pathway (Type 2 apoptosis) selectively in cancer cells, a mechanism that can be key to developing successful anti-cancer therapy. This work reports mathematical modeling and computer simulations to explore the mechanisms for cancer cell apoptosis. The results indicate that a combination of low probability Bid-Bax type reaction along with overexpressed reactant molecules allows specific killing of cancer cells. Low-probability activation of Bax also emerges as a basis for inherent cell-to-cell variability in apoptotic activation. Variations in Bcl-2 to Bax ratio within a cancer cell population can further affect intrinsic fluctuations generated due to the stochastic Bid-Bax reaction. Such heterogeneity in apoptosis resistance can also provide a mechanism for the origin of cells with higher tumorigenic potential (cancer stem-like cells). The implications of our results for cancer therapy, such as in minimizing stochastic fluctuations in cancer cell death, are discussed.

<div>Abstract<p><b>Purpose:</b> The selective killing of tumor cells is an important strategy for cancer therapeutics. The aim of this study was to develop a novel antitumor agent that is safe for normal cells with the ability to selectively target cancer cells.</p><p><b>Experimental Design:</b> On the basis of quantitative structure–activity relationship, we synthesized a novel polyphenol conjugate (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP-23). We evaluated the effect of DPP-23 on proliferation, cell cycle, and apoptosis in various tumor cells. We also assessed molecular targets of DPP-23 using genome-wide expression profiling by DNA microarray and real-time PCR array systems.</p><p><b>Results:</b> DPP-23 effectively inhibited the growth of cancer cells <i>in vitro</i> and <i>in vivo</i> (xenografts in Balb/c nude mice). At a molecular level, DPP-23 targeted the unfolded protein response (UPR) in the endoplasmic reticulum (ER) through the production of reactive oxygen species (ROS) in cancer cells, but not in normal cells, resulting in selective killing of tumor cells via caspase-dependent apoptosis.</p><p><b>Conclusions:</b> The selective generation of ROS in cancer cells could be an attractive strategy for the selective killing of cancer cells, while maintaining negligible cytotoxicity to normal cells. DPP-23 represents a promising novel therapeutic agent for the selective production of ROS in cancer cells. <i>Clin Cancer Res; 20(16); 4302–13. ©2014 AACR</i>.</p></div>