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 pPAKThr212 followed by late increase in pPAKThr423. 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 pPAKThr423 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 pPAKThr423, 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

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.This work is funded by a grant (MPP 320133) from the American University of Beirut to Dr. Ali H. Eid

Klinisches Potenzial von Thymoquinon im kolorektalen Karzinom: Identifizierung molekularer Targets und Wirksamkeit in Kombination Therapie

Being the most common cancer in Germany and having an important impact worldwide, colorectal cancer (CRC) has been studies extensively to achieve an appropriate therapy for this endemic disease. This PhD thesis aimed to study the effect of the naturally derived drug Thymoquinone (TQ) against CRC. In a first place we investigated the effect of TQ in combination along with traditional chemotherapy. Therefore we explored the ability of TQ, when combined with 5-Fluorouracil (5-FU) to chemosensitize apoptosis-resistant, p53-mutant HT29 colorectal cancer cells. The combined treatment induced a remarkable reduction in cell viability, an increased apoptosis rate, and an inhibition of anchorage-independent growth. Cell cycle analysis suggests that, upon combined treatment, the heavily damaged HT29 cells exit the S-phase and enter the M-phase due to the deficient G2/M checkpoint control. We observed as well cytoskeletal reorganization after single and combination treatment. Due to the high number of multinucleated cells we hypothesize that mitotic catastrophe could be at the origin of the cell death observed after combining TQ and 5-FU. In concordance with in vitro data, in vivo combination of TQ and 5-FU led to a significant reduction of relative tumor size and a higher survival rate. In a second place we examined the possible role of TQ in targeted therapy. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. By the mean of a kinome array, we identified 50 proteins showing a ≥2 fold-upregulation after TQ treatment. In silico analysis revealed several candidates belonging to the AKT-MEK-ERK1/2 pathway. The oncogenic p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced early increase of pERK1/2 and triggered the 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, thus abolishing the accessibility of Thr212 to ERK2. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with the kinase-dead dominant negative K299R mutant. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEKERK1/ 2 signaling and disrupting its prosurvival function. In conclusion, TQ revealed to be beneficial in both combinational and targeted therapies against CRC. Combined with 5-FU, TQ possess the potential to overcome drug resistance in HT29 cells. On the other hand, we showed that TQ can affect classical oncogenic targets belonging to known cancer related signaling pathways. For the first time we show that TQ directly binds to a kinase like PAK1 and induces changes in its conformation and its scaffold function. Finally we strongly recommend the combination of TQ with available therapies in future anti-CRC treatments.Das kolorektale Karzinom ist eine der häufigsten bösartigen Erkrankungen in Deutschland und weltweit von großer Bedeutung. Es wurden umfangreiche Studien durchgeführt, um eine optimale Therapie für diese endemische Erkrankung zu entwickeln. Ziel dieser Dissertation ist die Erforschung der Auswirkung des natürlichen Wirkstoffes Thymoquinon (TQ) auf das kolorektale Karzinom. Zunächst interessierten wir uns für die Wirkung von TQ in Kombination mit traditioneller Chemotherapie. Daher untersuchten wir in der kolorektalen Karzinomzelllinie HT29, welche apoptoseresistent ist und eine p53 Mutation aufweist, die Fähigkeit einer Chemosensibilisierung von TQ in Kombination mit 5-Fluoruracil (5-FU). Diese Wirkstoffkombination induzierte eine bemerkenswerte Reduktion des Zellüberlebens, eine gesteigerte Apoptoserate und eine Hemmung der Koloniebildung im soft agar colony Assay. Die Zellzyklusanalyse ergab, dass die unter Wirkstoffkombination schwer geschädigten HT29-Zellen aufgrund einer Störung am G2/M Kontrollpunkt aus der S-Phase direkt in die M-Phase eintreten. Gleichermaßen konnten wir eine Reorganisation des Zytoskeletts sowohl nach Einzelgabe als auch nach Wirkstoffkombination beobachten. Wegen der hohen Anzahl vielkerniger Zellen nehmen wir an, dass die mitotische Katastrophe den Grund für den Zelltod darstellt, der nach der Wirkstoffkombination TQ und 5-FU beobachtet wurde. In Übereinstimmung mit den Ergebnissen in vitro, ergab die Wirkstoffkombination TQ und 5-FU im Xenograft-Mausmodell eine signifikante Verminderung der relativen Tumorgröße und eine höhere Überlebensrate. Es wurde bereits gezeigt, dass TQ die Aktivität von Kinasen beeinflusst. In Anbetracht dessen, dass Kinasen vielversprechende Kandidaten für eine Targeted- Therapie darstellen, haben wir die durch TQ regulierten Kinasen genauer untersucht. Anhand eines Kinom-Arrays konnten wir 50 Proteine identifizieren, die eine mehr als 2-fache Heraufregulation nach TQ-Gabe aufzeigen. In silico Analysen ergaben, dass mehrere Kandidaten zum AKT-MEK-ERK1/2 Signalweg gehören. Die onkogene p21 PAK1 (protein (Cdc42/Rac)-activated kinase 1) zeichnet sich dabei als ein interessantes TQ-Target ab. PAK1 besitzt 2 Aminosäurereste, die zu unterschiedlichen Zeitpunkten phosphoryliert werden. Dadurch ergibt sich ein spezifisches Kinase-Profil: zuerst entsteht vermehrt pPAKThr212 dann, zu einem späteren Zeitpunkt, pPAKThr423. TQ induziert weiter eine vermehrte Phosphorylierung von ERK1/2 (pERK1/2) und fördert die Komplexbildung von ERK1/2-PAK1. Die strukturelle Modellierung dieses Phänomens ergab, dass TQ tatsächlich in der Umgebung von Thr212 bindet, was von einer dramatischer Veränderung in der Proteinkonformation begleitet wird. Somit kann ERK2 nicht mehr an der Phosphorylierungsstelle Thr212 binden. Die Transfektion der Zellen mit der Mutante T212A (nicht phosphorylierbar) zeigt einen Anstieg von pPAKThr423 und eine erhöhte Apoptoserate. In Zellen, die mit der Mutante K299R (Kinaseaktivität ausgeschaltet) transfiziert wurden, beobachteten wir ebenfalls eine erhöhte Apoptoserate. Die strukturelle Modellierung erklärt, dass TQ auch mit der Kinasedomäne interagiert und dadurch ihr Zusammenspiel mit PAKThr423 stört und somit das über den MEK-ERK1/2 Signalweg verbundene Überlebenssignal hemmt. Abschließend lässt sich sagen, dass TQ sowohl in Kombination mit traditioneller Chemotherapie als auch als Targeted-Therapie bei der Behandlung von kolorektalen Karzinomen gute Aussichten auf Erfolg hat. In Kombination mit 5-FU besitzt TQ das Potential, die Wirkstoffresistenz der HT29-Zellen zu überwinden. Weiterhin haben wir gezeigt, dass TQ klassische onkogene Targets, die zu bekannten tumorassozierten Signalwegen gehören, beeinflussen kann. Zum ersten Mal konnten wir nachweisen, dass TQ direkt an eine Kinase wie PAK1 bindet und dadurch Änderungen in ihrer Struktur und Gerüstfunktion induziert. Letztendlich empfehlen wir die Kombination von TQ mit bereits klinisch verfügbaren Therapien für das kolorektale Karzinom

Retinoids and Reactive Oxygen Species in Cancer Cell Death and Therapeutics

Retinoids are a large family of structurally related natural and synthetic molecules that exhibit a variety of effects in embryogenesis and adult life. The parent compound of retinoids is vitamin A which was first discovered for its impact on vision and later on cell proliferation, cell differentiation, and cell death. Clinically, retinoids were used for six decades in cancer management as they exhibit antitumor effects such as inhibiting cellular proliferation, inducing apoptosis, modulating cell cycle, and cell differentiation. Initially, early research focused on the mechanism of action of retinoids by their binding to retinoic acid receptors. However, recently accumulated evidence points that retinoids can act independent of the retinoid receptor signaling pathway for example by inducing stress, changes in redox balance, and reactive oxygen species (ROS) generation, and modulation of pathways that do not require binding of transcription factors to retinoic acid responsive DNA sequences. This review focuses on the mechanism of action of natural as well as synthetic retinoids, in particular, on the generation of ROS in in vitro and in vivo tumor models. A better understanding of the effects of retinoids on cellular redox balance and oxidative stress will impact future strategies of cancer management and therapy.Scopu

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

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

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 pPAKThr212 followed by late increase in pPAKThr423. 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 pPAKThr423 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 pPAKThr423, 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

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

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. 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. 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.</p