The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

Mannose Inhibits the Pentose Phosphate Pathway in Colorectal Cancer and Enhances Sensitivity to 5-Fluorouracil Therapy.

Colorectal cancer (CRC) is one of the leading cancers and causes of death in patients. 5-fluorouracil (5-FU) is the therapy of choice for CRC, but it exhibits high toxicity and drug resistance. Tumorigenesis is characterized by a deregulated metabolism, which promotes cancer cell growth and survival. The pentose phosphate pathway (PPP) is required for the synthesis of ribonucleotides and the regulation of reactive oxygen species and is upregulated in CRC. Mannose was recently reported to halt tumor growth and impair the PPP. Mannose inhibitory effects on tumor growth are inversely related to the levels of phosphomannose isomerase (PMI). An in silico analysis showed low PMI levels in human CRC tissues. We, therefore, investigated the effect of mannose alone or in combination with 5-FU in human CRC cell lines with different p53 and 5-FU resistance statuses. Mannose resulted in a dose-dependent inhibition of cell growth and synergized with 5-FU treatment in all tested cancer cell lines. Mannose alone or in combination with 5-FU reduced the total dehydrogenase activity of key PPP enzymes, enhanced oxidative stress, and induced DNA damage in CRC cells. Importantly, single mannose or combination treatments with 5-FU were well tolerated and reduced tumor volumes in a mouse xenograft model. In summary, mannose alone or in combination with 5-FU may represent a novel therapeutic strategy in CRC.

Lessons learned from the discovery and development of the sesquiterpene lactones in cancer therapy and prevention.

INTRODUCTION: Sesquiterpene lactones (SLs) are one of the most diverse bioactive secondary metabolites found in plants and exhibit a broad range of therapeutic properties . SLs have been showing promising potential in cancer clinical trials, and the molecular mechanisms underlying their anticancer potential are being uncovered. Recent evidence also points to a potential utility of SLs in cancer prevention. AREAS COVERED: This work evaluates SLs with promising anticancer potential based on cell, animal, and clinical models: Artemisinin, micheliolide, thapsigargin dehydrocostuslactone, arglabin, parthenolide, costunolide, deoxyelephantopin, alantolactone, isoalantolactone, atractylenolide 1, and xanthatin as well as their synthetic derivatives. We highlight actionable molecular targets and biological mechanisms underlying the anticancer therapeutic properties of SLs. This is complemented by a unique assessment of SL mechanisms of action that can be exploited in cancer prevention. We also provide insights into structure-activity and pharmacokinetic properties of SLs and their potential use in combination therapies. EXPERT OPINION: We extract seven major lessons learned and present evidence-based solutions that can circumvent some scientific limitations or logistic impediments in SL anticancer research. SLs continue to be at the forefront of cancer drug discovery and are worth a joint interdisciplinary effort in order to leverage their potential in cancer therapy and prevention.

The Pentose Phosphate Pathway in Cancer: Regulation and Therapeutic Opportunities.

BACKGROUND: Tumorigenesis is associated with deregulation of nutritional requirements, intermediary metabolites production, and microenvironment interactions. Unlike their normal cell counterparts, tumor cells rely on aerobic glycolysis, through the Warburg effect. SUMMARY: The pentose phosphate pathway (PPP) is a major glucose metabolic shunt that is upregulated in cancer cells. The PPP comprises an oxidative and a nonoxidative phase and is essential for nucleotide synthesis of rapidly dividing cells. The PPP also generates nicotinamide adenine dinucleotide phosphate, which is required for reductive metabolism and to counteract oxidative stress in tumor cells. This article reviews the regulation of the PPP and discusses inhibitors that target its main pathways. Key Message: Exploiting the metabolic vulnerability of the PPP offers potential novel therapeutic opportunities and improves patients' response to cancer therapy.

Terpenoids' anti-cancer effects: focus on autophagy.

Terpenoids are the largest class of natural products, most of which are derived from plants. Amongst their numerous biological properties, their anti-tumor effects are of interest for they are extremely diverse which include anti-proliferative, apoptotic, anti-angiogenic, and anti-metastatic activities. Recently, several in vitro and in vivo studies have been dedicated to understanding the 'terpenoid induced autophagy' phenomenon in cancer cells. Light has already been shed on the intricacy of apoptosis and autophagy relationship. This latter crosstalk is driven by the delicate balance between activating or silencing of certain proteins whereby the outcome is expressed via interrelated signaling pathways. In this review, we focus on nine of the most studied terpenoids and on their cell death and autophagic activity. These terpenoids are grouped in three classes: sesquiterpenoid (artemisinin, parthenolide), diterpenoids (oridonin, triptolide), and triterpenoids (alisol, betulinic acid, oleanolic acid, platycodin D, and ursolic acid). We have selected these nine terpenoids among others as they belong to the different major classes of terpenoids and our extensive search of the literature indicated that they were the most studied in terms of autophagy in cancer. These terpenoids alone demonstrate the complexity by which these secondary metabolites induce autophagy via complex signaling pathways such as MAPK/ERK/JNK, PI3K/AKT/mTOR, AMPK, NF-kB, and reactive oxygen species. Moreover, induction of autophagy can be either destructive or protective in tumor cells. Nevertheless, should this phenomenon be well understood, we ought to be able to exploit it to create novel therapies and design more effective regimens in the management and treatment of cancer.

Novel therapeutic strategies for spinal osteosarcomas.

At the dawn of the third millennium, cancer has become the bane of twenty-first century man, and remains a predominant public health burden, affecting welfare and life expectancy globally. Spinal osteogenic sarcoma, a primary spinal malignant tumor, is a rare and challenging neoplastic disease to treat. After the conventional therapeutic modalities of chemotherapy, radiation and surgery have been exhausted, there is currently no available alternative therapy in managing cases of spinal osteosarcoma. The defining signatures of tumor survival are characterised by cancer cell ability to stonewall immunogenic attrition and apoptosis by various means. Some of these biomarkers, namely immune-checkpoints, have recently been exploited as druggable targets in osteosarcoma and many other different cancers. These promising strides made by the use of reinvigorated immunotherapeutic approaches may lead to significant reduction in spinal osteosarcoma disease burden and corresponding reciprocity in increase of survival rates. In this review, we provide the background to spinal osteosarcoma, and proceed to elaborate on contribution of the complex ecology within tumor microenvironment giving arise to cancerous immune escape, which is currently receiving considerable attention. We follow this section on the tumor microenvironment by a brief history of cancer immunity. Also, we draw on the current knowledge of treatment gained from incidences of osteosarcoma at other locations of the skeleton (long bones of the extremities in close proximity to the metaphyseal growth plates) to make a case for application of immunity-based tools, such as immune-checkpoint inhibitors and vaccines, and draw attention to adverse upshots of immune-checkpoint blockers as well. Finally, we describe the novel biotechnique of CRISPR/Cas9 that will assist in treatment approaches for personalized medication.

Combination of 5-fluorouracil and thymoquinone targets stem cell gene signature in colorectal cancer cells.

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle stem cell regulatory pathways WNT/ß-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ß-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of ß-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.

Understanding CAM Use in Lebanon: Findings from a National Survey.

The main objective of this study was to identify predictors of Complementary and Alternative Medicine (CAM) use in Lebanon. Data for this study were drawn from a national survey conducted among Lebanese adults (n=1500). A modified version of the Social Behavioral Model (SBM) was used to understand CAM use in the study population. In this version, predisposing factors included sociodemographic characteristics (age, gender, education, and employment) and Push and Pull factors. Additionally, enabling resources included income, and medical need encompassed presence of chronic disease and perceived health status. Simple and multiple logistic regressions were used to examine the predictors of CAM use in the study population. Results of the multiple logistic regression showed that younger and older adults were less likely to use CAM as compared to middle-aged respondents. The Push factor "dissatisfaction with conventional medicine" was associated with higher odds of CAM use. For three of the six Pull factors, compared to participants who strongly disagreed, those who had a tendency of taking care of one's health were more likely to use CAM. Income and presence of chronic disease were also associated with higher odds of CAM use. The findings of this study affirmed the utility of the SBM in explaining the use of CAM and proposed a new version of this model, whereby the Push and Pull factors are integrated within the predisposing factors of this model.

Synthesis of Novel Hybrids of Thymoquinone and Artemisinin with High Activity and Selectivity Against Colon Cancer.

Colorectal cancer causes 0.5 million deaths each year. To combat this type of cancer the development of new specific drug candidates is urgently needed. In the present work seven novel thymoquinone-artemisinin hybrids with different linkers were synthesized and tested for their in vitro anticancer activity against a panel of various tumor cell lines. The thymoquinone-artesunic acid hybrid 7 a, in which both subunits are connected via an ester bond, was found to be the most active compound and selectively decreased the viability of colorectal cancer cells with an IC50 value of 2.4 µm (HCT116) and 2.8 µm (HT29). Remarkably, hybrid 7 a was up to 20-fold more active than its parent compounds (thymoquinone and artesunic acid), while not affecting nonmalignant colon epithelial HCEC cells (IC50 >100 µm). Moreover, the activity of hybrid 7 a was superior to that of various 1:1 mixtures of thymoquinone and artesunic acid. Furthermore, hybrid 7 a was even more potent against both colon cancer cell lines than the clinically used drug 5-fluorouracil. These results are another excellent proof of the hybridization concept and confirm that the type and length of the linker play a crucial role for the biological activity of a hybrid drug. Besides an increase in reactive oxygen species (ROS), elevated levels of the DNA-damage marker γ-H2AX were observed. Both effects seem to be involved in the molecular mechanism of action for hybrid 7 a in colorectal cancer cells.

Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells.

The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53(+/+) /p21(+/+) and null for p53(+/+) /p21(-/-) or p53(-/-) /p21(+/+) , we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study.

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer.

BACKGROUND: Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. METHODS: Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ's effects on PAK1 scaffold function. RESULTS: Of the 104 proteins identified, 50 were upregulated ≥ 2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAK(Thr212) followed by late increase in pPAK(Thr423). TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAK(Thr423) and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAK(Thr423), finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo. CONCLUSIONS: Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.

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.

Thymoquinone induces apoptosis in malignant T-cells via generation of ROS.

We show that HTLV-1 negative leukemia cells are more sensitive to TQ due to higher levels of drug-induced reactive oxygen species (ROS). PreG1 population in HTLV-1 negative Jurkat and CEM was higher than HTLV-1 transformed HuT-102 and MT-2 cells. Peripheral blood mononuclear cells were more resistant. Hoechst staining indicated more features of apoptosis, namely nuclear blebs and shrunken nuclei in HuT-102 than Jurkat. A greater depletion of the antioxidant enzyme glutathione occurred in Jurkat, which consequently led to an increase in ROS, loss of mitochondrial membrane potential, cytochrome c release, activation of caspases 3 and 9, and cleavage of PARP. Treatment with z-VAD-fmk partially reversed TQ-induced apoptosis, suggesting a caspase-dependent mechanism. N-acetyl cysteine prevented apoptosis providing evidence that cell death is ROS-dependent. Catalase prevented apoptosis to a lesser extent than NAC. In summary, TQ induces apoptosis in adult T cell leukemia/lymphoma by decreasing glutathione and increasing ROS, and levels of ROS underlie the differential cellular response to TQ. Our data suggest a potential therapeutic role for TQ in sensitizing HTLV-I-negative T-cell lymphomas.

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.