Comparative cytotoxicity of chelidonine and homochelidonine, the dimethoxy analogues isolated from Chelidonium majus L. (Papaveraceae), against human leukemic and lung carcinoma cells

doi:10.1016/j.phymed.2016.01.001

Phytomedicine

Volume 23, Issue 3, 15 March 2016, Pages 253-266

https://doi.org/10.1016/j.phymed.2016.01.001 Get rights and content

Abstract

Background

The search for new anticancer compounds is a crucial element of natural products research.

Purpose

In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described.

Methods

The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, clonogenic assay and for monitoring microtubule stability.

Results

We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells.

Conclusion

Collectively, the data indicate that chelidonine and homochelidonine are potent inducers of cell death in cancer cell lines, highlighting their potential relevance in leukemic cells.

Graphical abstract

Introduction

Alkaloids are a rich as well as an important source for searching for pharmacologically active drugs in anticancer treatment. Isoquinoline alkaloids, which currently number more than 2500 members, are isolated mainly from plants of subclasses Magnoliidae and Ranunculidae (Blaschek et al. 2010). Isoquinoline alkaloids are also widely spread in the poppy plant family (Papaveraceae) (Ziegler and Facchini 2008). The core isoquinoline nucleus may be present in plants belonging to the Papaveraceae as such, or as a structural moiety integrated into alkaloids classified as pavines, isopavines, benzophenanthridines, rhoeadines, papaverrubines, protopines, phthalideisoquinolines, protoberberines, aporphines and morphinans (Preininger 1985). Among these isoquinoline alkaloids, the benzophenanthridines have shown a promising cytostatic potential (Mansoor et al. 2013).

Chelidonine and homochelidonine (Fig. 1), B/C-cis-11-hydroxyhexahydrobenzo[c]phenanthridine alkaloids classified as partially hydrogenated-type congeners, were isolated and described as the main natural constituents of Chelidonium majus L. by Schmidt and Selle in the early 20th century (Šimánek, 1985, Panzer et al., 2001). At first, chelidonine received the most attention. Chelidonine was described as analgesic, antispasmodic, antibacterial, antiviral, antifungal, antioxidant, acetylcholinesterase and butyrylcholinesterase inhibitory (Colombo and Bosisio, 1996, Hiller et al., 1998, Gilca et al., 2010, Cahlíková et al., 2010). Later, chelidonine also exhibited cytotoxic, antiproliferative and apoptosis-inducing activity in diverse cancer cell lines (Kemény-Beke et al., 2006, Kaminskyy et al., 2008, Paul et al., 2012). It has recently been described that chelidonine causes an increase in DNA damage assayed by γH2AX in response to 1 and 2 h of treatment given a concentration of 3 µg/ml in A-375 and A-375-p53DD malignant melanoma cells (Hammerová et al. 2011). Chelidonine also showed the ability to overcome the multi-drug resistance (MDR) of different cancer cell lines through interaction with ABC-transporters, CYP3A4 and GST, by the induction of apoptosis accompanied by an activation of caspases -3, -8, and -6/9 (El-Readi et al. 2013). Interestingly, much less is known about the biological effects of homochelidonine. Similarly to chelidonine, homochelidonine was found to possess morphine-like properties, acetylcholinesterase and butyrylcholinesterase inhibitory activity (Weber and Hecker, 1978, Cahlíková et al., 2010). Contrary to chelidonine, evidence for cytotoxic and apoptosis-inducting activity of homochelidonine is currently missing.

Although evidence of the cytotoxic activities on alkaloids isolated from Chelidonium majus L. of the Papaveraceae family is currently growing, reports indicating biological differences between the main representatives are still very limited. Therefore, the aim of the present study was to characterize the cytotoxic and apoptosis inducing capacity of naturally occurring homochelidonine extracted from Chelidonium majus L. We evaluated the influence of homochelidonine in comparison with chelidonine. The cytotoxicity was evaluated against a mini-panel of human leukemic (MOLT-4, Jurkat, HL-60 and HEL 92.1.7), lymphoma (Raji and U-937), quiescent peripheral blood mononuclear (PBMCs) and healthy primary (MRC-5 and WI-38) cells. Human leukemic T-cells MOLT-4 (p53 wild-type) and Jurkat (p53 deficient) were further used for the evaluation of the viability, proliferation, apoptosis, cell cycle progression, mitotic block and expression of selected cell death- and/or cell cycle arrest-associated proteins. Hypotriploid non-small human lung adenocarcinoma cells A549 (p53 wild-type) were used for real-time continuous analysis of proliferation using the xCELLigence system and for clonogenic survival assay. A549 cells were also employed as a model for indirect immunofluorescence with an anti-β-tubulin antibody due to a lower nuclear-cytoplasmic ratio compared to lymphoblast cells.

Section snippets

Cell cultures and culture conditions

The experiments were carried out with the Jurkat (p53 mutant E6.1), MOLT-4 (p53 wild-type), Raji (p53 mutant), HL-60 (p53-deficient), U-937 (p53 mutant), HEL 92.1.7 (p53 wild-type), A549 (p53 wild-type), MRC-5 (primary human lung fibroblast) and WI-38 (primary human lung fibroblast) cell lines from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Jurkat and Raji cells were propagated in RPMI 1640 medium supplemented with 10% foetal bovine serum, 2 mM l-glutamine, 1 mM pyruvate, 10

Cytotoxicity screening of the homochelidonine and chelidonine towards human blood cancer and healthy cells

The homochelidonine and chelidonine were subjected to cytotoxic evaluation against 6 human blood cancer cell lines (Jurkat, MOLT-4, HL-60, Raji, U-937 and HEL 92.1.7), 2 human primary cell lines (MRC-5 and WI-38) and quiescent human PBMCs employing XTT assay. Table 1 demonstrates that blood cancer cells were more sensitive to the cytotoxic activity of homochelidonine (with exception of HEL 92.1.7. and U-937 cells) and chelidonine than the PBMCs. Moreover, PBMCs maintained higher viability at

Discussion

Our experiments focused on comparing the effects of chelidonine and naturally occurring chelidonine-dimethoxy analogue with open dioxole ring homochelidonine in parallel on leukemic T-cell lymphoblasts with different p53 status. The results presented here showed that chelidonine and homochelidonine affected the proliferation and viability of the human leukemic cells examined. Homochelidonine displayed preferentially concentration-dependent activity, which was not the case of chelidonine in the

Conflict of interest

The authors declare that there is no conflict of interest to reveal.

Acknowledgements

The authors would like to thank Ing. Ondrej Sedlak and Mr. Pavel Rozkosny (Nikon spol. s r.o., Czech Republic) for collaboration on the confocal microscopy and super-resolution microscopy imaging experiments. We also wish to thank Ivana Fousova for her skilful technical assistance. This study was financially supported by the ROUTER CZ.1.07/2.3.00/30.0058 Programme of the University of Pardubice and the PRVOUK P37/01 Programme of Charles University in Prague. Radim Havelek is co-financed by the

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From the previous studies this is evident that chelidonine has anticancer effects through several different mechanisms and it might act as a potential anticancer agent [36]. Chelidonine induces mitotic catastrophe and apoptotic-like death of SGC-7901 cells [37], inhibits tubulin polymerization and cell cycle progression in leukemic, lung carcinoma and HeLa cells [38,39], potentiates apoptosis in HeLa cells by p38-p53 and AKT/PI3 kinase pathways [40], induces caspase-dependent or caspase-independent cell apoptosis by G2/M arrest on glioblastoma cells [41], reduces telomerase activity by downregulation of hTERT and senescence induction in HepG2 cells [42], inhibits the invasion and migration of MDA-MB-231 breast cancer cells via suppressing the formation of integrin-linked kinase/PINCH/α-parvin complex [43], and restrains MCF-7 breast cancer cells growth via multiple mechanisms [44]. Chelidonine also suppresses epithelial mesenchymal transition and aggrandizes the sensitivity of lenvatinib on HCC cells [45], and it is identified as an ATP-competitive inhibitor of STK19 kinase activity and potentiates apoptosis through inhibition of NRAS signaling [46].
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Comparative cytotoxicity of chelidonine and homochelidonine, the dimethoxy analogues isolated from Chelidonium majus L. (Papaveraceae), against human leukemic and lung carcinoma cells

Abstract

Background

Purpose

Methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Cell cultures and culture conditions

Cytotoxicity screening of the homochelidonine and chelidonine towards human blood cancer and healthy cells

Discussion

Conflict of interest

Acknowledgements

Pharmacol. Res.

Phytomedicine

J. Dermatol. Sci.

Biochem. Biophys. Res. Commun.

Methods

Eur. J. Pharmacol.

Cell Biol. Int.

Cancer Lett.

Phytomedicine

Chem. Biol. Interact.

Eur. J. Cell Biol.

Hagers Enzyklopädie der Arzneistoffe und Drogen, HagerROM

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Chelidonium majus(Papaveraceae)

Nat. Prod. Commun.