Advanced fractionation of tropical healing plants used against inflammation

Während des Lebens erkrankt durchschnittlich eine von drei Personen an Krebs (Pecorino, 2008) und mehr als 50 % der betroffenen Personen sterben an der Krankheit (Stewart, et al., 2003). Allein diese beiden Fakten zeigen die große Notwendigkeit für Medikamente gegen Krebs auf. 60 % der Medikamente zur Tumorbehandlung stammen von Pflanzen ab, womit Pflanzen eine wichtige Grundlage für die Entwicklung dieser Medikamente bilden (Cragg, et al., 2005). Aus diesem Grund wurden die zwei ethnomedizinischen Pflanzen, die von den Maya in Guatemala verwendet werden, auf ihre anti-neoplastische Wirkung hin untersucht. Ethnomedizinische Pflanzen sind deshalb so wichtig in der Krebsforschung, da diese Pflanzen über Jahrhunderte hinweg gegen verschiedene Krankheiten verwendet wurden (Shoeb, 2006). Die zwei in dieser Studie untersuchten Pflanzen, P. odorata and S. spinosa, wurden ausgewählt, da sie ursprünglich zur Behandlung von Entzündungen verwendet wurden und bei Entzündungen und Krebs ähnliche Signalwege angesprochen werden (Kundu, et al., 2008). Das Dichlormethan-Extrakt der P. odorata ist bekannt dafür anti-neoplastisch zu wirken, wie Gridling et al. (2009) und Bauer et al. (2010) bereits beschrieben. Deshalb wurde die „Bio-Assay Guided Fractionation“ der P. odorata mit dem Dichlormethan-Extrakt begonnen. Nach vier bzw. fünf Fraktionierungsschritten mit Vakuum-Flüssigkeitschromatographie bzw. normaler Säulenchromatographie, wurden zwei sehr aktive Fraktionen erhalten; F4.6.3 und F5.3.6.7. Zwischen den jeweiligen Fraktionierungsschritten wurde die pro-apoptotische und anti-proliferative Aktivität der Fraktionen an HL-60 Zellen getestet. Die zytotoxischen Effekte dieser zwei Fraktionen waren erheblich, in der Konzentration von 10 µg/ml rief F4.6.3 nach 48 Stunden eine Apoptoserate von 100 % in HL-60 Zellen hervor, F5.3.6.7 von 90 %. Mittels Western Blots wurden diese Effekte weiter analysiert. Hier zeigte F4.6.3 einen etwas stärkeren Effekt auf die γH2AX Aktivierung, welche auf eine höhere Anzahl an DNA-Doppelstrangbrüchen hinweist. Als Konsequenz wurde die Caspase-Kaskade in Gang gesetzt, und der Zellzyklus durch die Phosphorylierung von Chk2 und Cdc2A angehalten. Im Gegensatz zu F4.6.3 zeigte F5.3.6.7 einen größeren Einfluss auf Mobilitätsproteine. F5.3.6.7 verminderte die Expression von Paxillin und ROCK-1 und hemmte die Phosphorylierung von MYPT, welche allesamt für eine Einschränkung der Zellmobilität stehen und damit den Weg für eine Metastasierung erschweren. Die getrocknete Wurzel der zweiten ethnomedizinischen Pflanze, S. spinosa, wurde zuerst gemahlen und anschließend mit Lösungsmitteln mit steigender Polarität extrahiert. Danach wurden Proliferations- und Apoptose-Assays durchgeführt und das aktivste Extrakt - das Methanol-Extrakt - einer Detannifizierung unterzogen. Nach dem Detannifizierungs-Prozess war die anti-neoplastische Aktivität in der Wasser-Methanol-Fraktion zu finden, in welcher auch die Tannine enthalten sein sollten. Die Tannine konnten jedoch nicht detektiert werden. Die Proliferation von HL-60 Zellen wurde durch die neu erhaltene Wasser-Methanol-Fraktion nach 48 Stunden in einer Konzentration von 60 µg/ml um 40 % gehemmt. Apoptose wurde in der Konzentration von 120 µg/ml nach 48 Stunden in 95 % der HL-60 Zellen induziert. Auf Grund dieses Ergebnisses wurden Onkoproteine, bzw. Proteine die am Zellzyklus oder der Apoptose beteiligt sind, mittels Western Blots näher untersucht. Die Fraktion hatte vor allem großen Einfluss auf Onkogene wie beispielsweise Stat3 und c-Myc. Die Phosphorylierung von Stat3 wurde durch die Wasser-Methanol-Fraktion vermindert, ebenso wie die c-Myc-Expression, welche beide als Indikatoren für abnormales Zellwachstum bekannt sind. Außerdem führte die Fraktion zu Unterbrechungen des Zellzyklus durch verminderte Expression von Cdc25A nach 24 Stunden und erhöhte Expression von p(Ser177)Cdc25A und p21. Ebenfalls wurden Indikatoren für Apoptose durch die Fraktion von S. spinosa beeinflusst. Die γH2AX Expression stieg und zusätzlich wurde die Caspase-Kaskade in Gang gesetzt, bei der es durch Aktivierung von Caspase 8 und 9 zur Spaltung von Caspase 3 kommt. Caspase 3 ist bekannt für die Induktion von Apoptose und für die Aktivierung von PARP, welches ebenfalls ein Apoptose Indikator ist. Zusammenfassend kann gesagt werden, dass beide untersuchten Pflanzen deutliche anti-neoplastische Effekte in HL-60 Zellen zeigten und deshalb untermauern, dass ethnomedizinische Pflanzen, die seit Jahrhunderten als Medikament gegen Entzündung in Verwendung sind, eine gute Grundlage für die Forschung nach neuen Krebsmedikamenten bilden.The facts that one out of three persons is affected by cancer during their lifetime (Pecorino, 2008) and that more than 50 % of them die from the disease (Stewart, et al., 2003) show that there is a substantial need for anti-cancer drugs. Plants have been a source of effective drugs for the treatment of cancers, and generally over 60 % of anti-cancer drugs originate from natural sources (Cragg, et al., 2005). For this reason two ethnomedical plants, used by the Mayas of Guatemala, were investigated towards their anti-neoplastic activity. Ethnomedical plants have a large impact in cancer research, because those natural products have been administered as a therapy against several diseases for hundreds of years (Shoeb, 2006). The two plants investigated in this study, P. odorata and S. spinosa, are originally used against inflammation. Reason for the investigation of plants with anti-inflammatory effects is that both, inflammation and cancer up-regulate similar signalling pathways (Kundu, et al., 2008). The dichloromethane extract of P. odorata is already known to contain an anti-neoplastic activity, as described by Gridling et al. (2009) and Bauer et al. (2010). Therefore the bio-assay guided fractionation of P. odorata started with the dichloromethane extract. After four, respectively five steps of fractionation using vacuum liquid chromatography or column chromatography with in-between testing the pro-apoptotic and anti-proliferative effect on HL-60 cells, the fractionation process resulted in two very active fractions; F4.6.3 and F5.3.6.7. The two fractions were then further analysed by western blot analysis with focus on cytotoxic effects. In the concentration of 10 µg/ml both fractions had strong cytotoxic effects. F4.6.3 induced apoptosis by 100 % in HL-60 cells after 48 hours; F5.3.6.7 induced an apoptosis rate of 90 %. The western blot analysis revealed that F4.6.3 was the fraction with the stronger γH2AX activation, indicating DNA-double-strand breaks. For this reason the fraction showed also the activation of the caspase cascade and inhibition of the cell cycle progression by phosphorylation of Chk2 and Cdc25A. In contrast, F5.3.6.7 had a stronger impact on the cell mobility proteins like decreasing the expression of paxillin and ROCK-1. This and the complete inhibition of pMYPT are indicative for decreased cell mobility, decreasing the chance for metastasis. The air-dried root of the second ethnomedical plant from Guatemala, S. spinosa, was first milled and then extracted by solvents with increasing polarity. Afterwards proliferation and apoptosis assays were conducted. The most active extract, the methanol extract was then subjected to detannification. The anti-proliferative and pro-apoptotic activity was found in the water-methanol layer, which should also include the tannins that however have not been detectible. The extract inhibited proliferation up to 40 % in the concentration of 60 µg/ml and induced apoptosis up to 95 % in the concentration of 120 µg/ml after 48 hours of incubation. Proteins relating cell cycle progression, apoptosis and oncoproteins were investigated by western blotting. The fraction especially had a great impact on oncogenes. It decreased pStat3 as well as c-Myc, which are all indicators for abnormal cell growth. Further the water-methanol fraction led to cell cycle disturbances leading to growth arrest by decreasing the expression of Cdc25A after 24 hours and increasing the level of p(Ser177)Cdc25A and p21. Apoptosis indicating proteins like γH2AX and the caspase cascade including caspase 8, 9 and in the end the cleavage of caspase 3, which itself results in a PARP cleavage were up-regulated by the extract of S. spinosa. In summary, both investigated plants clearly showed anti-neoplastic effects on HL-60 cells, and therefore confirm that ethnomedical plants, used against inflammation for hundreds of years are suitable to discover novel anti-cancer drugs

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT

The human RAD5 homologs, HLTF and SHPRH, have separate functions in DNA damage tolerance dependent on the DNA lesion type

Helicase-like transcription factor (HLTF) and SNF2, histone-linker, PHD and RING finger domain-containing helicase (SHPRH), the two human homologs of yeast Rad5, are believed to have a vital role in DNA damage tolerance (DDT). Here we show that HLTF, SHPRH and HLTF/SHPRH knockout cell lines show different sensitivities towards UV-irradiation, methyl methanesulfonate (MMS), cisplatin and mitomycin C (MMC), which are drugs that induce different types of DNA lesions. In general, the HLTF/SHPRH double knockout cell line was less sensitive than the single knockouts in response to all drugs, and interestingly, especially to MMS and cisplatin. Using the SupF assay, we detected an increase in the mutation frequency in HLTF knockout cells both after UV- and MMS-induced DNA lesions, while we detected a decrease in mutation frequency over UV lesions in the HLTF/SHPRH double knockout cells. No change in the mutation frequency was detected in the HLTF/SHPRH double knockout cell line after MMS treatment, even though these cells were more resistant to MMS and grew faster than the other cell lines after treatment with DNA damaging agents. This phenotype could possibly be explained by a reduced activation of checkpoint kinase 2 (CHK2) and MCM2 (a component of the pre-replication complex) after MMS treatment in cells lacking SHPRH. Our data reveal both distinct and common roles of the human RAD5 homologs dependent on the nature of DNA lesions, and identified SHPRH as a regulator of CHK2, a central player in DNA damage response

APIM-Mediated REV3L-PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.publishedVersion© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)