Novel Therapeutic Strategies for Cutaneous T-Cell-Lymphoma and Its Effect on Apoptosis Regulation

Cutaneous T-cell lymphomas (CTCL) are a relatively rare disease with an estimated 3-4 new cases per year per million population in Europe and yet have been classified as se-rious due to lifelong morbidity and permanent sustained impairment of quality of life. Cur-rent therapies are often inadequate or lacking durable efficacy. Signaling pathways that inducing cell death are important for both suppression of tumorigenesis and efficacy of the antitumor therapy. However, the mechanisms leading to cell death in CTCL are cur-rently poorly understood. The aim of this study was to identify new therapeutic strategies to induce apoptosis in CTCL cells and to investigate the apoptotic signaling pathways in CTCL cells at the mo-lecular level. Four CTCL cell lines (HH, HuT-78, MyLa, SeAx) were treated with different effectors and subsequently analyzed by flow cytometry to quantify apoptosis, ROS pro-duction as well as loss of mitochondrial membrane potential and for regulation of the apoptotic signaling pathways by Westernblot. In this study, three therapeutic strategies were identified. A proapoptotic effect of ingenol mebutate PEP005 was demonstrated in two of four CTCL cell lines. The mechanism was associated with PKCδ (protein kinase C-delta) activation and subsequent activation of proapoptotic caspase-3. The resistant cell lines were characterized by certain molecu-lar characteristics such as expression of p53 and p21, as well as increased expression of the antiapoptotic proteins c-FLIP and XIAP, which could thus serve as candidates for stratification of patients. BH3 (Bcl-2 homology domain 3) mimetics represented the sec-ond group of compounds studied. The investigation on Mcl-1 inhibitor S63845 showed that two out of four CTCL cell lines were highly sensitive. The two cell lines with only low sensitivity to S63845, on the other hand, were characterized by high sensitivity to ABT-263 and ABT-737, which are known as specific inhibitors of Bcl-2, Bcl-w, and Bcl-xL. The sensitivity to S63845 was found to be correlated with the decreased expression of Bcl-2 and Bcl-xL and most importantly, with the absence of Bcl-w expression. Bcl-w could therefore be used as a biomarker for sensitivity of CTCL to S63845. The third strategy was based on the indirubin derivative DKP-071, which induced apoptosis depending on ROS induction. These three studied effectors could potentially be used as new therapeutic strategies for CTCL. Further analyses are required to validate the current findings.Die kutanen T-Zell-Lymphome (CTCL) sind mit geschätzten 3-4 Neuerkrankungen pro Jahr und einer Million Einwohner in Europa eine relativ seltene Erkrankung, die jedoch aufgrund der lebenslangen Morbidität und dauerhaft nachhaltigen Beeinträchtigung der Lebensqualität als schwerwiegend eingestuft wurde. Aktuelle Therapien sind häufig unzureichend oder haben keine dauerhafte Wirksamkeit. Signalwege, die den Zelltod induzieren, sind sowohl für die Unterdrückung der Tumorentstehung als auch für die erfolgreiche Umsetzung einer Antitumortherapie wichtig. Jedoch sind die Prozesse, die zum Zelltod beim CTCL führen, derzeit noch unzureichend verstanden. Ziel der Arbeit war es, neue Therapieansätze zur Apoptoseinduktion in CTCL-Zellen zu identifizieren und die apoptotischen Signalwege in CTCL-Zellen auf molekularer Ebene zu untersuchen. Vier CTCL-Zelllinien (HH, HuT-78, MyLa, SeAx) wurden mit verschiedenen Effektoren behandelt und anschließend mittels Durchflusszytometrie zur Quantifizierung von Apoptose, ROS Produktion sowie Verlust des mitochondrialen Membranpotentials sowie mittels Westernblot die Regulation der apoptotischen Signalwege untersucht. In dieser Arbeit konnten drei therapeutische Strategien indentifiziert werden. Eine proapoptotische Wirkung des Ingenolmebutats PEP005 wurde in zwei von vier CTCL Zelllinien nachgewiesen. Der Mechanismus war mit der Aktivierung der PKCδ (Protein Kinase C-delta) sowie der darauffolgenden Aktivierung der proapoptotischen Caspase-3 verbunden. Die resistenten Zelllinien zeichneten sich durch bestimmte molekulare Merk male wie die Expression von p53 und p21 sowie durch eine verstärkte Expression der antiapoptotischen Faktoren c-FLIP und XIAP aus, die somit als Kandidaten für eine Stratifizierung von Patienten dienen könnten. Die zweite untersuchte Substanzgruppe stellten BH3-Mimetika dar. Dies sind Inhibitoren für antiapoptotische Bcl-2-Proteine, die strukturelle Ähnlichkeit zur BH3-Domäne (Bcl-2 homology domain 3) besitzen. Die Untersuchung des Mcl-1-Inhibitors S63845 zeigte, dass zwei von vier CTCL-Zelllinien hohe Sensitivität besaßen, während die zwei Zelllinien mit geringer Sensitivität für S63845 andererseits durch eine hohe Sensitivität gegenüber den für Bcl-2, Bcl-w und Bcl-xL spezifischen Inhibitoren ABT-263 und ABT-737 charakterisiert waren. Die Sensitivität gegenüber S63845 korrelierte mit einer verminderten Expression von Bcl-2 und Bcl-xL sowie einer fehlenden Bcl-w-Expression. Bcl-w könnte da her als Biomarker für die S63845-Sensitivität des CTCLs eingesetzt werden. ie dritte Strategie beruhte auf dem Indirubin-Derivat DKP-071, die Apoptose in Abhängigkeit der ROS Produktion induzierte. Diese drei untersuchten Effektoren könnten als neue therapeutische Strategien für das CTCL genutzt werden. Weitere Untersuchungen sind erforderlich, um die aktuellen Ergebnisse zu validieren

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKC delta, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKC delta appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting

Sensitivity of Cutaneous T-Cell Lymphoma Cells to the Mcl-1 Inhibitor S63845 Correlates with the Lack of Bcl-w Expression

Long-term, curative treatment of cutaneous T-cell lymphomas (CTCL) remains a major challenge. Therapy resistance is often based on apoptosis deficiency, and may depend on antiapoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1. For their targeting, several antagonists have been generated, which mimic the Bcl-2 homology domain 3 (BH3 mimetics). As dysregulation and overexpression of Mcl-1 has been reported in CTCL, the use of Mcl-1 inhibitors appears as an attractive strategy. Here, we investigated the effects of the selective Mcl-1 inhibitor S63845 in a series of four CTCL cell lines, in comparison to ABT-263 and ABT-737 (inhibitors of Bcl-2, Bcl-xL and Bcl-w). In two cell lines (HH, HuT-78), S63845 resulted in significant apoptosis induction, decrease in cell viability, loss of mitochondrial membrane potential and caspase activation, while two other cell lines (MyLa, SeAx) remained completely resistant. An inverse correlation was found, as S63845-resistant cells were highly sensitive to ABT-263/-737, and S63845-sensitive cells showed only moderate sensitivity to ABTs. Combinations of S63845 and ABT-263 partially yielded synergistic effects. As concerning Bcl-2 protein expression, weaker Mcl-1 expression was found in S63845-resistant MyLa and SeAx, while for Bcl-2 and Bcl-xL, the lowest expression was found in the highly sensitive cell line HH. The most striking difference between S63845-resistant and -sensitive cells was identified for Bcl-w, which was exclusively expressed in S63845-resistant cells. Thus, CTCL may be efficiently targeted by BH3 mimetics, providing the right target is preselected, and Bcl-w expression may serve as a suitable marker

Key Role of Reactive Oxygen Species (ROS) in Indirubin Derivative-Induced Cell Death in Cutaneous T-Cell Lymphoma Cells

Cutaneous T-cell lymphoma (CTCL) may develop a highly malignant phenotype in its late phase, and patients may profit from innovative therapies. The plant extract indirubin and its chemical derivatives represent new and promising antitumor strategies. This first report on the effects of an indirubin derivative in CTCL cells shows a strong decrease of cell proliferation and cell viability as well as an induction of apoptosis, suggesting indirubin derivatives for therapy of CTCL. As concerning the mode of activity, the indirubin derivative DKP-071 activated the extrinsic apoptosis cascade via caspase-8 and caspase-3 through downregulation of the caspase antagonistic proteins c-FLIP and XIAP. Importantly, a strong increase of reactive oxygen species (ROS) was observed as an immediate early effect in response to DKP-071 treatment. The use of antioxidative pre-treatment proved the decisive role of ROS, which turned out upstream of all other proapoptotic effects monitored. Thus, reactive oxygen species appear as a highly active proapoptotic pathway in CTCL, which may be promising for therapeutic intervention. This pathway can be efficiently activated by an indirubin derivative