Therapeutic potential of thymoquinone in combination therapy against cancer and cancer stem cells.

The long-term success of standard anticancer monotherapeutic strategies has been hampered by intolerable side effects, resistance to treatment and cancer relapse. These monotherapeutic strategies shrink the tumor bulk but do not effectively eliminate the population of self-renewing cancer stem cells (CSCs) that are normally present within the tumor. These surviving CSCs develop mechanisms of resistance to treatment and refuel the tumor, thus causing cancer relapse. To ensure durable tumor control, research has moved away from adopting the monotreatment paradigm towards developing and using combination therapy. Combining different therapeutic modalities has demonstrated significant therapeutic outcomes by strengthening the anti-tumor potential of monotreatment against cancer and cancer stem cells, mitigating their toxic adverse effects, and ultimately overcoming resistance. Recently, there has been growing interest in combining natural products from different sources or with clinically used chemotherapeutics to further improve treatment efficacy and tolerability. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has gained great attention in combination therapy research after demonstrating its low toxicity to normal cells and remarkable anticancer efficacy in extensive preclinical studies in addition to its ability to target chemoresistant CSCs. Here, we provide an overview of the therapeutic responses resulting from combining TQ with conventional therapeutic agents such as alkylating agents, antimetabolites and antimicrotubules as well as with topoisomerase inhibitors and non-coding RNA. We also review data on anticancer effects of TQ when combined with ionizing radiation and several natural products such as vitamin D3, melatonin and other compounds derived from Chinese medicinal plants. The focus of this review is on two outcomes of TQ combination therapy, namely eradicating CSCs and treating various types of cancers. In conclusion, the ability of TQ to potentiate the anticancer activity of many chemotherapeutic agents and sensitize cancer cells to radiotherapy makes it a promising molecule that could be used in combination therapy to overcome resistance to standard chemotherapeutic agents and reduce their associated toxicities.

Emerging Cytotoxic Alkaloids in the Battle against Cancer: Overview of Molecular Mechanisms.

Considered as the second deadliest disease globally, cancer has captured the attention of researchers who have been trying with perseverance to decode its hidden aspects, to find new prognosis methods, and to develop better and more effective treatments. Plants have continuously offered an excess of unique secondary metabolites with remarkable biological applications. Alkaloids, one of the most abundant metabolites, constitute a large conglomerate of basic heterocyclic nitrogen-containing natural compounds which are normally produced by plants as toxic substances. Out of the 27,000 different alkaloids, more than 17,000 have displayed diversified pharmacological properties including anticancer activities. These metabolites have been classified either according to their chemical structures or their taxonomic origin. None of the researched alkaloids have been classified according to their molecular mechanism of action against cancer. In fact, only a fraction of the tremendous number of anticancer alkaloids has been copiously mentioned in journals. Here, we aim to provide a summary of the literature on some of the promising anticancer alkaloids that have not been well discussed previously and to classify them according to their molecular mechanisms of action. This review will provide a better understanding of the anticancer mechanisms of these promising natural products that are a rich reservoir for drug discovery.

Cell death mechanisms of plant-derived anticancer drugs: beyond apoptosis.

Despite remarkable progress in the discovery and development of novel cancer therapeutics, cancer remains the second leading cause of death in the world. For many years, compounds derived from plants have been at the forefront as an important source of anticancer therapies and have played a vital role in the prevention and treatment of cancer because of their availability, and relatively low toxicity when compared with chemotherapy. More than 3000 plant species have been reported to treat cancer and about thirty plant-derived compounds have been isolated so far and have been tested in cancer clinical trials. The mechanisms of action of plant-derived anticancer drugs are numerous and most of them induce apoptotic cell death that may be intrinsic or extrinsic, and caspase and/or p53-dependent or independent mechanisms. Alternative modes of cell death by plant-derived anticancer drugs are emerging and include mainly autophagy, necrosis-like programmed cell death, mitotic catastrophe, and senescence leading to cell death. Considering that the non-apoptotic cell death mechanisms of plant-derived anticancer drugs are less reviewed than the apoptotic ones, this paper attempts to focus on such alternative cell death pathways for some representative anticancer plant natural compounds in clinical development. In particular, emphasis will be on some promising polyphenolics such as resveratrol, curcumin, and genistein; alkaloids namely berberine, noscapine, and colchicine; terpenoids such as parthenolide, triptolide, and betulinic acid; and the organosulfur compound sulforaphane. The understanding of non-apoptotic cell death mechanisms induced by these drugs would provide insights into the possibility of exploiting novel molecular pathways and targets of plant-derived compounds for future cancer therapeutics.

Thymoquinone: fifty years of success in the battle against cancer models.

Thymoquinone (TQ), the main active constituent of black seed essential oil, exhibits promising effects against inflammatory diseases and cancer. TQ, modulates signaling pathways that are key to cancer progression, and enhances the anticancer potential of clinical drugs while reducing their toxic side effects. Considering that TQ was isolated 50 years ago, this review focuses on TQ's chemical and pharmacological properties and the latest advances in TQ analog design and nanoformulation. We discuss our current state of knowledge of TQ's adjuvant potential and in vivo antitumor activity and highlight its ability to modulate the hallmarks of cancer.

Differential growth inhibitory effects of highly oxygenated guaianolides isolated from the Middle Eastern indigenous plant Achillea falcata in HCT-116 colorectal cancer cells.

Medicinal plants play a crucial role in traditional medicine and in the maintenance of human health worldwide. Sesquiterpene lactones represent an interesting group of plant-derived compounds that are currently being tested as lead drugs in cancer clinical trials. Achillea falcata is a medicinal plant indigenous to the Middle Eastern region and belongs to the Asteraceae family, which is known to be rich in sesquiterpene lactones. We subjected Achillea falcata extracts to bioassay-guided fractionation against the growth of HCT-116 colorectal cancer cells and identified four secotanapartholides, namely 3-ß-methoxy-isosecotanapartholide (1), isosecotanapartholide (2), tanaphallin (3), and 8-hydroxy-3-methoxyisosecotanapartholide (4). Three highly oxygenated guaianolides were isolated for the first time from Achillea falcata, namely rupin A (5), chrysartemin B (6), and 1ß, 2ß-epoxy-3ß,4α,10α-trihydroxyguaian-6α,12-olide (7). These sesquiterpene lactones showed no or minor cytotoxicity while exhibiting promising anticancer effects against HCT-116 cells. Further structure-activity relationship studies related the bioactivity of the tested compounds to their skeleton, their lipophilicity, and to the type of functional groups neighboring the main alkylating center of the molecule.

Synergistic anticancer activities of the plant-derived sesquiterpene lactones salograviolide A and iso-seco-tanapartholide.

We have previously shown that the two sesquiterpene lactones, salograviolide A (Sal A) and iso-seco-tanapartholide (TNP), isolated from the Middle Eastern indigenous plants Centaurea ainetensis and Achillea falcata, respectively, possess selective antitumor properties. Here, we aimed to assess the anticancer effects of the separate compounds and their combination, study their potential to generate reactive oxygen species (ROS), and investigate their underlying antitumor mechanisms in human colon cancer cell lines. Cells were treated with Sal A and TNP alone or in combination, and cell viability, cell cycle profile, apoptosis, ROS generation and changes in protein expression were monitored. Sal A and TNP in combination caused 80% decrease in HCT-116 and DLD-1 cell viability versus only 25% reduction when the drugs were used separately. The antitumor mechanism involved triggering ROS-dependent apoptosis as well as disruption of the mitochondrial membrane potential. Further studies showed that apoptosis by the Sal A and TNP combination was caspase-independent and that ERK, JNK and p38 of the serine/threonine MAPKs signaling pathway were involved in the cell death mechanism. Taken together, our data suggest that the combination of Sal A and TNP may be of therapeutic interest against colon cancer.

A journey under the sea: the quest for marine anti-cancer alkaloids.

The alarming increase in the global cancer death toll has fueled the quest for new effective anti-tumor drugs thorough biological screening of both terrestrial and marine organisms. Several plant-derived alkaloids are leading drugs in the treatment of different types of cancer and many are now being tested in various phases of clinical trials. Recently, marine-derived alkaloids, isolated from aquatic fungi, cyanobacteria, sponges, algae, and tunicates, have been found to also exhibit various anti-cancer activities including anti-angiogenic, anti-proliferative, inhibition of topoisomerase activities and tubulin polymerization, and induction of apoptosis and cytotoxicity. Two tunicate-derived alkaloids, aplidin and trabectedin, offer promising drug profiles, and are currently in phase II clinical trials against several solid and hematologic tumors. This review sheds light on the rich array of anti-cancer alkaloids in the marine ecosystem and introduces the most investigated compounds and their mechanisms of action.

Sage components enhance cell death through nuclear factor kappa-B signaling.

The sage components linalyl acetate (Ly) and alpha-terpineol (Te) exhibit synergistic anti-proliferative effects. We investigated the effects of Ly and Te on NF-kappaB signaling in HCT-116 colon cancer cells. Ly and Te combinations dose-dependently reduced HCT-116 viability at non-cytotoxic concentrations. Combination treatment induced 30%-60% increase in PreG1 through induction of apoptosis and necrosis. DNA binding assays revealed that combination treatment suppressed both basal and TNF-alpha-induced NF-kappaB activation. This suppression correlated with the inhibition of p65 nuclear translocation and IkappaB-alpha degradation. The lack of change in IKK expression levels or inhibition in IkappaB-alpha phosphorylation suggest the involvement of an IKK-independent mechanism. Ly and Te combination was found to downregulate the expression of NF-kappaB-regulated antiapoptotic and proliferative gene products. Separate treatments and drug combinations significantly decreased DNA binding activity of NF-kappaB which led to the potentiation of cell death induced by the colon cancer drugs oxaliplatin and 5-FU. These results indicate that Ly and Te anticancer activities are partly mediated through the suppression of NF-kappaB activation, suggesting their use in combination with chemotherapeutic agents to induce apoptosis.

Cell death by the quinoxaline dioxide DCQ in human colon cancer cells is enhanced under hypoxia and is independent of p53 and p21.

INTRODUCTION: We have shown that the radio sensitizer DCQ enhances sensitivity of HCT116 human colon cancer cells to hypoxia. However, it is not known whether the p53 or p21 genes influence cellular response to DCQ. In this study, we used HCT116 that are either wildtype for p53 and p21, null for p53 or null for p21 to understand the role of these genes in DCQ toxicity. METHODS: HCT116 cells were exposed to DCQ and incubated under normoxia or hypoxia and the viability, colony forming ability, DNA damage and apoptotic responses of these cells was determined, in addition to the modulation of HIF-1α and of p53, p21, caspase-2, and of the ataxia telangiectasia mutated (ATM) target PIDD-C. RESULTS: DCQ decreased colony forming ability and viability of all HCT116 cells to a greater extent under hypoxia than normoxia and the p21-/-cell line was most sensitive. Cells had different HIF-1α responses to hypoxia and/or drug treatment. In p53+/+, DCQ significantly inhibited the hypoxia-induced increases in HIF-1α protein, in contrast to the absence of a significant HIF-1α increase or modulation by DCQ in p21-/- cells. In p53-/- cells, 10 µM DCQ significantly reduced HIF-1α expression, especially under hypoxia, despite the constitutive expression of this protein in control cells. Higher DCQ doses induced PreG1-phase increase and apoptosis, however, lower doses caused mitotic catastrophe. In p53+/+ cells, apoptosis correlated with the increased expression of the pro-apoptotic caspase-2 and inhibition of the pro-survival protein PIDD-C. Exposure of p53+/+ cells to DCQ induced single strand breaks and triggered the activation of the nuclear kinase ATM by phosphorylation at Ser-1981 in all cell cycle phases. On the other hand, no drug toxicity to normal FHs74 Int human intestinal cell line was observed. CONCLUSIONS: Collectively, our findings indicate that DCQ reduces the colony survival of HCT116 and induces apoptosis even in cells that are null for p53 or p21, which makes it a molecule of clinical significance, since many resistant colon tumors harbor mutations in p53.

What made sesquiterpene lactones reach cancer clinical trials?

Sesquiterpene lactones (SLs) are plant-derived compounds often used in traditional medicine against inflammation and cancer. This review focuses on the chemical and biological properties of SLs that lead to enhanced anticancer and anti-inflammatory effects. The chemical properties comprise alkylating center reactivity, lipophilicity, and molecular geometry and electronic features. SLs in clinical trials are artemisinin, thapsigargin and parthenolide and many of their synthetic derivatives. These drugs are selective toward tumor and cancer stem cells by targeting specific signaling pathways, which make them lead compounds in cancer clinical trials.

Anti-colon cancer effects of Salograviolide A isolated from Centaurea ainetensis.

The antitumor activity of extracts of Centaurea ainetensis (C. ainetensis), a plant endemic to Lebanon, was investigated in human colon carcinoma cells. At concentrations that were non-cytotoxic to normal human intestinal epithelial cells, the crude extract inhibited the proliferation of a host of colon-derived cancer cells. The crude extract effect was then investigated in HCT-116 (p53+/+) cells, most sensitive to treatment and was found to cause apoptosis, increase the Bax/Bcl-2 ratio, p53 and p21 protein levels and reduce cyclin B1 proteins. In vivo, the crude extract injected intraperitoneally before the subcutaneous injection of the carcinogen 1,2-dimethylhydrazine, drastically reduced the number of tumors and decreased the mean size of aberrant crypt foci. Further bioassay-guided fractionation of the crude extract resulted in the identification of the bioactive molecule Salograviolide A, a Sesquiterpene Lactone, to which the growth inhibition in colon cancer was linked. Salograviolide A, at non-cytotoxic concentrations to normal human intestinal cells, reduced the growth of colon cancer cell lines. Salograviolide A induced growth inhibition and resulted in an increased preG1 phase and presumably apoptosis induction which was further confirmed by TUNEL. These data support the testing of the C. ainetensis extract and its bioactive molecule, Salograviolide A, in colon cancer treatment.

Purified salograviolide A isolated from centaurea ainetensis causes growth inhibition and apoptosis in neoplastic epidermal cells.

Many of the best-selling anticancer drugs are plant-derived. We tested for the anticancer properties of extracts isolated from Centaurea ainetensis, a plant species endemic to Lebanon and which is often used in folk medicine. We performed bioassay-guided fractionation of Centaurea ainetensis extracts using a panel of normal and neoplastic murine cells to identify a component that is associated with antitumor activities. Among several compounds that were fractionated, the sesquiterpene lactone, Salograviolide A, was identified and found to exert the most significant growth inhibitory effects on neoplastic cells. At concentrations that were non-cytotoxic to primary keratinocytes, Centaurea ainetensis crude extract and Salograviolide A preferentially inhibited the proliferation of papilloma and squamous cell carcinoma (SCC) cell lines without significantly affecting the growth of normal cells. Flow cytometric analysis of DNA content indicated that the inhibition of cell proliferation by Centaurea ainetensis crude extract and Salograviolide A was due to G0/G1 cell cycle arrest and increased pre-G0/G1, respectively. The increase in pre-G0/G1, and presumably apoptosis induction, in Salograviolide A-treated keratinocytes was confirmed by DNA Hoechst staining. Western blot analysis and electrophoretic mobility shift assay showed that both the crude extract and the isolated molecule differentially modulated key cell cycle and apoptotic regulators as well as NF-kappaB signaling. Salograviolide A-induced growth inhibition in neoplastic cells was mediated by the accumulation of reactive oxygen species (ROS) highlighting a potent oxidant role of this molecule. These studies suggest the potential therapeutic effects of Centaurea ainetensis, and its component, Salograviolide A, against epidermal squamous cell carcinogenesis.

Onopordum cynarocephalum induces apoptosis and protects against 1,2 dimethylhydrazine-induced colon cancer.

The potential chemopreventive properties of the crude extract of Onopordum cynarocephalum were evaluated. Growth inhibition was investigated in FHs74Int human normal intestinal cells and ModeK mouse normal intestinal cell line and in two human colon cancer cells HCT-116 (p53+/+) and HT-29 (p53+/-). The extract was not cytotoxic to FHs74Int cells at concentrations 2-fold higher than the IC50 of HCT-116 cells. The extract inhibited dose-dependently the growth of HCT-116 cells (IC50=0.18 mg/ml) to a greater extent than HT-29 cells (IC50=1.8 mg/ml). The p53 wild-type HCT-116 cells were more sensitive than p53 mutant HT-29 cells to the pro-apoptotic effects of the plant extract; five times lower concentrations were needed to induce apoptosis in HCT-116 cells. Apoptosis induction by the extract was associated with an increase in the expression of pro-apoptotic proteins such as p53 and Bax, and a significant inhibition of the anti-apoptotic protein Bcl-2. Significant decrease in cyclin D1 protein and increase in p21 protein was observed in extract-treated HCT-116 cells. In vivo, the crude extract injected intra-peritoneally reduced the number of tumors by 64% (p<0.0001) and decreased the mean size of aberrant crypt foci in the DMH model of colon cancer. These data collectively suggest that O. cynarocephalum has potential anti-colon cancer effects.

Thymoquinone: a promising anti-cancer drug from natural sources.

There has been growing interest in naturally occurring compounds with anti-cancer potential. Black seed is one of the most extensively studied plants. This annual herb grows in countries bordering the Mediterranean Sea and India. Thymoquinone (TQ) is the bioactive constituent of the volatile oil of black seed. It has been shown to exert anti-neoplastic and anti-inflammatory effects. The molecular pathways of TQ action are not clear. Nevertheless, TQ is known to induce apoptosis by p53-dependent and p53-independent pathways in cancer cell lines. Growth inhibition is associated with induction of cell cycle arrest. TQ also acts on the immune system by modulating the levels of inflammatory mediators. To date, the chemotherapeutic potential of TQ in the clinic has not been tested, but numerous studies have shown its promising anti-cancer effects in animal models. The combination of TQ with clinically used anti-cancer drugs has led to improvements in their therapeutic index and prevents non-tumor tissues from sustaining chemotherapy-induced damage.

5-Aza-cytidine is a potent inhibitor of DNA methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53-dependent mechanisms.

Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21(WAF1) and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G(2)/M-mediator cyclin B1. Co-incubation with pifithrin-alpha (PFT-alpha), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21(WAF1) expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16(INK4) expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-alpha reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21(WAF1) and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.

Protective effect of vitamin E on ultraviolet B light-induced damage in keratinocytes.

Ultraviolet (UV) B radiation is the most common environmental factor in the pathogenesis of skin cancer. Exposure of human skin to UVB radiation leads to the depletion of cutaneous antioxidants, the activation of nuclear factor kappa B (NF-kappaB), and programmed cell death (apoptosis). Although antioxidant supplementation has been shown to prevent UVB-induced photooxidative damage, its effect on components of cell signaling pathways leading to gene expression has not been clearly established. In the present study, the effect of the antioxidant vitamin, alpha-tocopherol (alpha-T), and its acetate analog, alpha-tocopherol acetate (alpha-TAc), on UVB-induced damage in primary and neoplastic mouse keratinocytes was investigated. The ability of both vitamins to modulate UVB-induced apoptosis and activation of the transcription factor NF-kappaB were studied. Treatment of normal and neoplastic mouse epidermal keratinocytes (308 cells) with 30-60 mJ/cm(2) UVB markedly decreased viable cell number and was accompanied by DNA fragmentation. When both vitamins were applied to cells at times before and after UVB radiation, a significant increase in the percentage of viable cells and concomitant decrease in the number of apoptotic cells was noted, with vitamin pretreatment providing a better protection than posttreatment. Simultaneous posttreatment of irradiated cells with alpha-TAc abolished the cytotoxic effects of UVB and restored cell viability to control levels. In addition, simultaneous posttreatment of irradiated cells with alpha-T reduced the number of apoptotic cells by half, indicating a synergistic effect of two such treatments compared with any single one. Flow cytometry analysis indicated that vitamin treatment suppressed both an increase in pre-G0 cells and a decrease in cycling cells by UVB exposure. In addition, NF-kappaB activation was detected 2 h after UV exposure and was maintained for up to 8 h. Pretreatment with vitamins significantly inhibited NF-kappaB activation at 4 and 8 h. These results indicate that vitamin E and its acetate analog can modulate the cellular response to UVB partly through their action on NF-kappaB activation. Thus, these antioxidant vitamins are potential drugs for the protection from or the reduction of UVB-associated epidermal damage.