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Differential effect of sanguinarine, chelerythrine and chelidonine on DNA damage and cell viability in primary mouse spleen cells and mouse leukemic cells
Cell Biology International 32 (2008) 271e277 www.elsevier.com/locate/cellbi
Differential effect of sanguinarine, chelerythrine and chelidonine on DNA damage and cell viability in primary mouse spleen cells and mouse leukemic cells Vitaliy Kaminskyy1, Kah-Wai Lin1, Yevhen Filyak, Rostyslav Stoika* Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences, Drahomanov Street 14/16, 79005 Lviv, Ukraine Received 14 May 2007; revised 1 August 2007; accepted 4 September 2007
Abstract Sanguinarine, chelerythrine and chelidonine are isoquinoline alkaloids derived from the greater celandine. They possess a broad spectrum of pharmacological activities. It has been shown that their anti-tumor activity is mediated via different mechanisms, which can be promising targets for anti-cancer therapy. We focused our study on the differential effects of these alkaloids upon cell viability, DNA damage effect and nucleus integrity in mouse primary spleen cells and mouse lymphocytic leukemic cells, L1210. Sanguinarine and chelerythrine produce a dosedependent increase in DNA damage and cytotoxicity in both primary mouse spleen cells and L1210 cells. Chelidonine did not show a significant cytotoxicity or damage DNA in both cell types, but completely arrested growth of L1210 cells. Examination of nuclear morphology revealed more cells with apoptotic features upon treatment with chelerythrine and sanguinarine, but not chelidonine. In contrast to primary mouse spleen cells, L1210 cells showed slightly higher sensitivity to sanguinarine and chelerythrine treatment. This suggests that cytotoxic and DNA damaging effects of chelerythrine and sanguinarine are more selective against mouse leukemic cells and primary mouse spleen cells, whereas chelidonine blocks proliferation of L1210 cells. The action of chelidonine on normal and tumor cells requires further investigation. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Alkaloids; Cytotoxicity; Apoptosis; Cancer cells
1. Introduction The greater celandine (Chelidonium majus L.) is a species of the poppy family (Papaveraceae L.) containing a variety of secondary metabolites, isoquinoline alkaloids (Fig. 1). This plant has been widely used in the treatment of various diseases (Colombo and Bosisio, 1996). Chelerythrine, sanguinarine and chelidonine are common isoquinoline alkaloids with a broad spectrum of pharmacological activities. For decades, their biological activities and clinical applications are extensively investigated. For many years, chelerythrine and * Corresponding author. Tel./fax: þ38 032 2612287. E-mail address: [email protected] (R. Stoika). 1 These authors contributed equally.
sanguinarine have been used in the treatment of gingival inflammation and supragingival plaque formation (Eley, 1999; Godowski et al., 1995). Ukrain, a semisynthetic thiophosphoric acid compound of chelidonine, has been shown to be a potential anti-cancer drug in clinical trial (Ernst and Schmidt, 2005). Studies demonstrated that these alkaloids possess anti-tumor, anti-inflammatory, anti-microbial, antifungal and anti-viral activities (Ahmad et al., 2000; Lenfeld et al., 1981; Vavreckova et al., 1996a,b; Walterova et al., 1995). Most notably, they possess anti-tumor activity which is mediated via different mechanisms that could be promising targets for anti-cancer therapy. Chelerythrine causes tumor cells death by initiating apoptosis through pathway directly targeting Bcl-2 family proteins (Chan et al., 2003; Malikova et al., 2006a). It is shown that
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.09.004
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2001). Chelidonine is a weak inhibitor of cell growth, but there is no evidence for selective cytotoxicity in normal, transformed and malignant cell lines (Panzer et al., 2001). Only a few studies on biological activity of chelidonine are available and further investigation is needed. To date, the differential cytotoxic effects of alkaloids in normal and cancer cells have been poorly studied (Ahmad et al., 2000; Malikova et al., 2006b). Current available chemotherapeutic agents have high toxicity towards normal cells in addition to tumor cells. Thus, searching for anti-tumor agents with minimal toxicity towards normal cells is of great value, and plant-derived products are in the great concern. Here we compared the effects of sanguinarine, chelerythrine and chelidonine upon the DNA damage, nucleus integrity and cytotoxicity in primary mouse spleen cells and mouse lymphocytic leukemic cell line, L1210. By comparing the effects of these alkaloids in normal and cancer cells, an in vitro animal model was established which provided important clues for their future applications. 2. Materials and methods 2.1. Extraction and purification of alkaloids
Fig. 1. Structure of the alkaloids.
Sanguinarine, chelerythrine and chelidonine were isolated and purified from roots of the greater celandine by Dr. Maxim Lootsik (see Kaminskyy et al., 2006). The alkaloids were dissolved in 50% ethanol and diluted in culture medium, taking into account that the final concentration of ethanol in culture medium did not exceed 0.1%.
2.2. Cell cultures
chelerythrine-mediated apoptosis occurred in various human tumor cell lines (Chan et al., 2003; Kemeny-Beke et al., 2006; Malikova et al., 2006b; Yamamoto et al., 2001). In addition, chelerythrine exhibited cytotoxic effect against radioresistant and chemoresistant squamous carcinoma cells with delayed tumor growth and mild toxicity in an animal model (Chmura et al., 2000). The usefulness of chelerythrine in combination with photodynamic therapy or ionizing radiation has been implicated (Chmura et al., 1997; Sausville et al., 1998). Sanguinarine induces cell cycle arrest and apoptosis via various pathways (Adhami et al., 2004; Malikova et al., 2006a). The involvement of mitochondrial pathway and Bcl-2 family proteins in apoptosis has been demonstrated (Ahmad et al., 2000). The inhibition of mitogen-activated protein kinase (MAPK) phosphatase, suppression of vascular endothelial growth factor (VEGF)-induced angiogenesis (Eun and Koh, 2004) and down-regulation of adhesion molecules (Tanaka et al., 2001) implicate sanguinarine as a potential agent against tumor progression and metastasis. Sanguinarine induces apoptosis in human squamous carcinoma without significant effect on normal human keratinocytes (Ahmad et al., 2000). A number of studies suggest that sanguinarine is a potential anti-tumor agent (Ding et al., 2002; Kemeny-Beke et al., 2006; Weerasinghe et al., 2001). Chelidonine induces cell cycle arrest and activation of stress-activated protein kinase/Jun kinase (SAK/JNK) pathway. It inhibits tubulin polymerization and disrupts microtubular structure in cells (Panzer et al.,
Mouse lymphocytic leukemic cells, L1210, were obtained from National Collection of Cell Lines at the Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv, Ukraine. Mouse spleen was removed, transferred to Petri dishes, and several small incisions were made on the side of spleen. By squeezing the spleen with phosphate-buffered saline, a single-cell suspension was produced. Yield and viability of cells were assessed by trypan blue exclusion method. Both cell types were cultured in RPMI 1640 medium (Sigma Chem. Co, USA) supplemented with 10% fetal bovine serum (Sigma Chem. Co, USA). Cells were maintained under standard cell culture conditions at 37 C with 5% CO2 in air at 100% humidity.
2.3. Alkaloid treatment Cells were cultured in 96-well plate at 2 105 per well. Chelerythrine, sanguinarine and chelidonine were added to the culture medium at different concentrations (0.5e8 mg/ml), and cells were incubated for 24 h at 37 C.
2.4. Determination of cell viability Cells’ viability was evaluated by trypan blue exclusion method. Cells were stained with 0.1% trypan blue for 10 min at room temperature. The number of live (transparent) and dead (blue) cells was counted in hemocytometric chamber under light microscope. Cell viability was denoted as percentage of live cells. Cell growth was calculated as percentage of increase in amount of cells in 24 h. The 50% lethal dosage (LD50) was determined from doseeresponse curves.
2.5. DNA comet assay at moderate alkaline pH For detection of DNA damage, comet assay at moderate alkaline pH was performed, as described by Kaminskyy et al. (2006). Briefly, cells were
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collected by centrifugation and resuspended in phosphate-buffered saline to final concentration of 3 105 cells/ml. The suspension was mixed with low melting point agarose and rapidly pipetted onto microscopic slides and covered with coverslips. Slides were immersed in lysis buffer (0.5 M EDTA, 2% lauryl-sarcosine, 0.3 mg/ml proteinase K, pH 7.5) for 60 min at 4 C, then for 20 h at 37 C. Slides were equilibrated by careful washing in electrophoresis buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.5) and electrophoresed at 0.6 V/cm for 25 min. The slides were stained with 2 mg/ml ethidium bromide in distilled water. DNA comets were analyzed using fluorescence microscope (LOMO, Russia) with barrier filter of 590 nm under magnification 200 and captured by digital camera. Comet images were analyzed using CometScore software (TriTek Corporation). The tail moment (tail length multiplied by the DNA density in the comet tail) was used as primary measurement for quantification of extent of DNA damage. A minimum of 100 cells were analyzed for each sample and data were transferred into Microsoft Excel software for analysis.
2.6. Morphology assessment of apoptosis Living unfixed cells were stained with fluorescent bis-benzimidazole dye, Hoechst 33342, at a final concentration of 1.5 mM in PBS. Cells were incubated for 20 min at 37 C. Nucleus morphology was analyzed using fluorescence microscope (LOMO, Russia) with 430 nm barrier filter (laser beam excitation at 360 nm) under 400 magnification. Images of random fields were captured by digital camera. Nucleus with chromatin condensation, nuclear fragmentation or apoptotic bodies was identified as apoptotic cells.
2.7. Statistical analysis All experiments were performed in triplicate. The Student’s t-test was used and P < 0.05 were considered as statistical significance. Statistical analyses were performed by Microsoft Excel.
3. Results 3.1. Cell viability The cytotoxic effect of alkaloids on cell growth was determined by trypan blue exclusion method in terms of cell viability (Figs. 2 and 3). Sanguinarine and chelerythrine exhibited cytotoxicity in dose-dependent manner in both cell types. Cells exposed to chelidonine only slightly decreased in viability, but showed complete arrest of cell growth at >4.6 mg/ml (Fig. 3). In primary mouse spleen cells, the LD50 after 24 h with sanguinarine and chelerythrine was 2.8 mg/ml and 4.6 mg/ml, respectively (Fig. 2A). In L1210 cells, the LD50 corresponding values were 1.9 mg/ml and 3.2 mg/ml, respectively (Fig. 2B). L1210 cells showed higher sensitivity to sanguinarine (>2 mg/ml) and chelerythrine (>4 mg/ml) treatment, compared to mouse spleen cells. There were no significant differences in cytotoxity in both cell types treated with chelidonine. 3.2. DNA comet assay In order to estimate DNA damage in cells exposed to alkaloids, comet assay at moderate alkaline pH was performed (Figs. 4 and 5). In both mouse spleen cells and L1210 cells, sanguinarine and chelerythrine showed a dose-dependent increase in the percentage of cells with DNA damage compared to control. Sanguinarine showed a statistically significant higher level of DNA damage at concentration >2 mg/ml in
Fig. 2. Effect of alkaloids on cell viability measured by trypan blue exclusion method in (A) mouse spleen cells and (B) mouse leukemic cells, L1210. Sanguinarine and chelerythrine exhibit cytotoxicity in dose-dependent manner in both mouse spleen cells and L1210 cells. Chelidonine showed only slight decrease in cell viability and did not show significant effect in dose-dependent manner, as compared to control. L1210 cells showed significant slightly higher sensitivity to sanguinarine and chelerythrine treatment, as compared to mouse spleen cells. The means values and standard deviations are shown.
spleen cells and >1 mg/ml in L1210 cells. Under treatment of chelerythrine, intensive DNA damage was seen at >4 mg/ ml in both spleen cells and L1210 cells. Chelidonine did not show any significant effect on DNA damage compared to control. L1210 cells showed significantly higher sensitivity to sanguinarine (4 mg/ml) and chelerythrine (4 mg/ml and 8 mg/ml) treatment compared to mouse spleen cells. In both cell types, sanguinarine at >8 mg/ml showed intensive DNA damage (data not presented). No significant differences were observed in both cell types after treatment of chelidonine. 3.3. Morphology assessment of apoptosis Hoechst 33342 dye penetrates the plasma membrane and stains DNA in the cells. The nuclei of apoptotic cells are
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4. Discussion
Fig. 3. Effect of alkaloids on cell growth in L1210 cells. Cells exposed to chelidonine exhibited complete arrest of cell growth at concentration >4.6 mg/ml.
characterized by chromatin condensation, nuclear fragmentation or presence of apoptotic bodies. We examined morphological changes of cell nuclei in order to assess our findings above (Fig. 6). In L1210 cells, the level of apoptotic features after treatment of sanguinarine and chelerythrine was consistent with cytotoxicity and DNA damage. Chelidonine showed atypical nuclear morphology in L1210 cells, which can be explained by its effect on cell growth and cell cycle arrest in mitosis.
The main goal of our study was to investigate the differences in cytotoxic activity of sanguinarine, chelerythrine and chelidonine in normal and cancer cells. We used primary mouse spleen cells and mouse lymphocytic leukemic cell line, L1210. Spleen cells are mainly composed of lymphocytes, thereby representing the normal counterpart of leukemic cells, while L1210 is mouse leukemia of lymphocytic origin with well characterized features that is commonly used as an in vitro leukemic model. Previous studies showed that chelerythrine and sanguinarine treatment of many cancer cell lines (Adhami et al., 2004; Ahmad et al., 2000; Chmura et al., 2000; KemenyBeke et al., 2006; Malikova et al., 2006b) and several primary cell culture and normal cell lines (Ahmad et al., 2000; Malikova et al., 2006b; Vavreckova et al., 1996a,b) resulted in dose-dependent decrease in cell viability. These studies are consistent with our results, showing that sanguinarine and chelerythrine decrease cell viability significantly in dosedependent manner in both mouse spleen cells and L1210 cells. We found that sanguinarine has significantly higher cytotoxicity than chelerythrine in both cell types, which agrees with a recent report on uveal melanoma cells (Kemeny-Beke et al., 2006). Our study showed that chelidonine only slightly decreased cell viability and did not show significant cytotoxicity as assessed by trypan blue exclusion. Previous studies have shown that cytotoxicity induced by chelidonine is much less
Fig. 4. Effect of alkaloids on DNA damage measured by comet assay in (A) mouse spleen cells þ sanguinarine; (B) L1210 cells þ sanguinarine; (C) mouse spleen cells þ chelerythrine; (D) L1210 cells þ chelerythrine; (E) mouse spleen cells þ chelidonine; and (F) L1210 cells þ chelidonine. Sanguinarine showed statistically significant higher level of DNA damage at concentration >2 mg/ml in spleen cells and >1 mg/ml in L1210 cells. Chelerythrine showed statistically significant higher level of DNA damage at concentration >4 mg/ml in spleen cells and L1210 cells. Chelidonine showed only slightly increase in DNA damage and did not show significant effect in DNA damage, as compared to control. L1210 cells showed significant higher sensitivity to sanguinarine (4 mg/ml) and chelerythrine (4 mg/ml and 8 mg/ml) treatment, as compared to mouse spleen cells (* indicates significant difference comparing to control value at P < 0.05 and þ indicates significant difference of both cell types).
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Fig. 5. Examples of DNA comet images (200). (A) Mouse spleen cells, control; (B) mouse spleen cells þ 4 mg/ml sanguinarine; (C) mouse spleen cells þ 8 mg/ml chelerythrine; (D) mouse spleen cells þ 8 mg/ml chelidonine; (E) L1210 cells, control; (F) L1210 cells þ 4 mg/ml sanguinarine; (G) L1210 cells þ 8 mg/ml chelerythrine; and (H) L1210 cells þ 8 mg/ml chelidonine. No tail detected in (A) and (E). Comets with small or no tail, which are similar to control (D, H). Comets with medium and long tail are showed in (B), (C), (F), and (G). Photos are representative of at least three independent experiments.
effective than by sanguinarine and chelerythrine (KemenyBeke et al., 2006; Vavreckova et al., 1996a,b). This is because the cytotoxic effects of sanguinarine and chelerythrine are attributed to the disruption of plasmatic membrane, which was detected by trypan blue exclusive method. Increased permeability of late apoptotic cells is the consequence of a compromised integrity of the plasma membrane. This corresponds with a previous study, which showed that human keratinocytes are susceptible for sanguinarine and chelerythrine in terms of plasma membrane integrity, resulting in a twofold increase in
the release of lactate dehydrogenase activity (Vavreckova et al., 1996b). On the other hand, chelidonine induced cell growth arrest (Panzer et al., 2001), which is consistent with our results. Comet assay was used to evaluate the level of DNA damage in individual cell. Sanguinarine and chelerythrine exhibited significant increase in DNA damage in dose-dependent manner in both mouse spleen cells and L1210 cells. Sanguinarine rendered higher levels of DNA damage than chelerythrine. Higher tail moments suggested more intensive DNA strand
Fig. 6. Examples of Hoechst 33342-stained cells (400). (A) Mouse spleen cells, control; (B) mouse spleen cells þ 4 mg/ml sanguinarine; (C) mouse spleen cells þ 8 mg/ml chelerythrine; (D) mouse spleen cells þ 8 mg/ml chelidonine; (E) L1210 cells, control; (F) L1210 cells þ 4 mg/ml sanguinarine; (G) L1210 cells þ 8 mg/ml chelerythrine; and (H) L1210 cells þ 8 mg/ml chelidonine. Apoptotic cells with chromatin condensation, nuclear fragmentation, or apoptotic bodies are showed in (B), (C), (F), and (G). Photos are representative of at least three independent experiments.
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breaks. DNA damaged cells possessed typical small head and large tail pattern, called ‘‘hedgehog cells’’, and revealed major DNA fragmentation resulting from late apoptotic events. Chelidonine did not show significant DNA damage in both cell types compared with controls. This observation supports our finding mentioned above, in which both cell types are sensitive to sanguinarine and chelerythrine treatment. Our results are consistent with a previous study in which human gingival fibroblasts and prostate cancer cell lines were used (Malikova et al., 2006b). Nuclear morphology was monitored by Hoechst 33342 staining. Apoptotic morphological features were more prominent in L1210 cells treated with chelerythrine and sanguinarine. Treatment of cells with chelidonine did not show a significant level of apoptosis, while chromatin disorganization occurred in L1210 cells. These findings are consistent with the observations described above. It is known that chelerythrine induced apoptosis in various normal and cancer cell types, such as human gingival fibroblast (Malikova et al., 2006b), squamous cell carcinoma (HNSCC) (Chmura et al., 2000), human leukemia (Chmura et al., 1996; Freemerman et al., 1996), human breast cancer (MCF-7), human colon carcinoma (Chan et al., 2003) and human uveal melanoma (OCM1) (Kemeny-Beke et al., 2006), while sanguinarine induced apoptosis in human erythroleukemia cells (K562) and human prostate carcinoma cells (Adhami et al., 2004). The detail mechanisms of chelerythrine and sanguinarine on apoptosis were extensively studied and well elucidated (Chan et al., 2003; Chaturvedi et al., 1997; Herbert et al., 1990; Hu et al., 2000; Vogt et al., 2005; Yu et al., 2000). Our result showed that L1210 cells are significantly more sensitive to sanguinarine and chelerythrine treatment compared with normal spleen cells in terms of disruption of plasmic membrane integrity and DNA damage. Chelidonine’s effect did not show significant difference between normal spleen cells and L1210 cells. These findings suggested that sanguinarine and chelerythrine are slightly more cytotoxic against cancer cells. Several studies support our findings. At micromolar concentrations, the effects on cell growth inhibition and apoptosis are significantly higher in human squamous carcinoma (A431) cells via induction of apoptosis, in comparison to normal human epidermal keratinocytes (Ahmad et al., 2000). Chelerythrine delays tumor growth in experimental model with relatively mild toxicity to animal (Chmura et al., 2000). In contrast, a recent study demonstrated that chelerythrine and sanguinarine exerted higher toxicity towards normal human gingival fibroblasts in comparison to prostate cancer cell lines (Malikova et al., 2006b). We propose that different cell types exert differential level of sensitivity to alkaloids treatment, although further experiments, both in vivo and in vitro, are crucial for evaluation of these alkaloids and their undesired toxic effect to normal cells. In conclusion, our study demonstrated that (1) in both normal spleen cells and L1210 leukemic cells, the effect of cytotoxicity or disruption of plasma membrane integrity, DNA damage and apoptosis are observed in dose-dependent manner after sanguinarine and chelerythrine treatment; (2) sanguinarine confers
higher level of cytotoxicity and DNA damage than chelerythrine, while chelidonine showed cell growth arrest with minimal cytotoxicity; and (3) L1210 cells were more sensitive to toxic effect of chelerythrine and sanguinarine treatment, when compared to normal mouse spleen cells. These results suggest that chelerythrine and sanguinarine possess slightly selective toxicity against cancer cells. However, further studies are needed to exploit their toxicity towards other normal cell types. The mechanism and signaling pathway of differential toxic effect of these alkaloids towards normal and cancer cells are of great interest for future studies, which are achievable by molecular and genomic approaches. Acknowledgements We thank Dr. Maxim Lootsik (Institute of Cell Biology, National Academy of Sciences, Lviv, Ukraine) for presenting alkaloids and Taras Soroka for his technical assistance in the experiments. References Adhami VM, Aziz MH, Reagan-Shaw SR, Nihal M, Mukhtar H, Ahmad N. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitorecycline cyclin-dependent kinase machinery. Mol Cancer Ther 2004;3:933e40. Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 2000;6:1524e8. Chan SL, Lee MC, Tan KO, Yang LK, Lee AS, Flotow H, et al. Identification of chelerythrine as an inhibitor of BclXL function. J Biol Chem 2003;278:20453e6. Chaturvedi MM, Kumar A, Darnay BG, Chainy GB, Agarwal S, Aggarwal BB. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kappaB activation, IkappaBalpha phosphorylation, and degradation. J Biol Chem 1997;272:30129e34. Chmura SJ, Nodzenski E, Crane MA, Virudachalam S, Hallahan DE, Weichselbaum RR, et al. Cross-talk between ceramide and PKC activity in the control of apoptosis in WEHI-231. Adv Exp Med Biol 1996;406:39e55. Chmura SJ, Mauceri HJ, Advani S, Heimann R, Beckett MA, Nodzenski E, et al. Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation. Cancer Res 1997;57:4340e7. Chmura SJ, Dolan ME, Cha A, Mauceri HJ, Kufe DW, Weichselbaum RR. In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo. Clin Cancer Res 2000;6:737e42. Colombo ML, Bosisio E. Pharmacological activities of Chelidonium majus L. (Papaveraceae). Pharmacol Res 1996;33:127e34. Ding Z, Tang SC, Weerasinghe P, Yang X, Pater A, Liepins A. The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death. Biochem Pharmacol 2002;63:1415e21. Eley BM. Antibacterial agents in the control of supragingival plaque e a review. Br Dent J 1999;186:286e96. Ernst E, Schmidt K. Ukrain e a new cancer cure? A systematic review of randomised clinical trials. BMC Cancer 2005;5:69. Eun JP, Koh GY. Suppression of angiogenesis by the plant alkaloid, sanguinarine. Biochem Biophys Res Commun 2004;317:618e24. Freemerman AJ, Turner AJ, Birrer MJ, Szabo E, Valerie K, Grant S. Role of c-jun in human myeloid leukemia cell apoptosis induced by pharmacological inhibitors of protein kinase C. Mol Pharmacol 1996;49:788e95.
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Differential effect of sanguinarine, chelerythrine and chelidonine on DNA damage and cell viability in primary mouse spleen cells and mouse leukemic cells Vitaliy Kaminskyy1, Kah-Wai Lin1, Yevhen Filyak, Rostyslav Stoika* Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences, Drahomanov Street 14/16, 79005 Lviv, Ukraine Received 14 May 2007; revised 1 August 2007; accepted 4 September 2007
Abstract Sanguinarine, chelerythrine and chelidonine are isoquinoline alkaloids derived from the greater celandine. They possess a broad spectrum of pharmacological activities. It has been shown that their anti-tumor activity is mediated via different mechanisms, which can be promising targets for anti-cancer therapy. We focused our study on the differential effects of these alkaloids upon cell viability, DNA damage effect and nucleus integrity in mouse primary spleen cells and mouse lymphocytic leukemic cells, L1210. Sanguinarine and chelerythrine produce a dosedependent increase in DNA damage and cytotoxicity in both primary mouse spleen cells and L1210 cells. Chelidonine did not show a significant cytotoxicity or damage DNA in both cell types, but completely arrested growth of L1210 cells. Examination of nuclear morphology revealed more cells with apoptotic features upon treatment with chelerythrine and sanguinarine, but not chelidonine. In contrast to primary mouse spleen cells, L1210 cells showed slightly higher sensitivity to sanguinarine and chelerythrine treatment. This suggests that cytotoxic and DNA damaging effects of chelerythrine and sanguinarine are more selective against mouse leukemic cells and primary mouse spleen cells, whereas chelidonine blocks proliferation of L1210 cells. The action of chelidonine on normal and tumor cells requires further investigation. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Alkaloids; Cytotoxicity; Apoptosis; Cancer cells
1. Introduction The greater celandine (Chelidonium majus L.) is a species of the poppy family (Papaveraceae L.) containing a variety of secondary metabolites, isoquinoline alkaloids (Fig. 1). This plant has been widely used in the treatment of various diseases (Colombo and Bosisio, 1996). Chelerythrine, sanguinarine and chelidonine are common isoquinoline alkaloids with a broad spectrum of pharmacological activities. For decades, their biological activities and clinical applications are extensively investigated. For many years, chelerythrine and * Corresponding author. Tel./fax: þ38 032 2612287. E-mail address: [email protected] (R. Stoika). 1 These authors contributed equally.
sanguinarine have been used in the treatment of gingival inflammation and supragingival plaque formation (Eley, 1999; Godowski et al., 1995). Ukrain, a semisynthetic thiophosphoric acid compound of chelidonine, has been shown to be a potential anti-cancer drug in clinical trial (Ernst and Schmidt, 2005). Studies demonstrated that these alkaloids possess anti-tumor, anti-inflammatory, anti-microbial, antifungal and anti-viral activities (Ahmad et al., 2000; Lenfeld et al., 1981; Vavreckova et al., 1996a,b; Walterova et al., 1995). Most notably, they possess anti-tumor activity which is mediated via different mechanisms that could be promising targets for anti-cancer therapy. Chelerythrine causes tumor cells death by initiating apoptosis through pathway directly targeting Bcl-2 family proteins (Chan et al., 2003; Malikova et al., 2006a). It is shown that
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2001). Chelidonine is a weak inhibitor of cell growth, but there is no evidence for selective cytotoxicity in normal, transformed and malignant cell lines (Panzer et al., 2001). Only a few studies on biological activity of chelidonine are available and further investigation is needed. To date, the differential cytotoxic effects of alkaloids in normal and cancer cells have been poorly studied (Ahmad et al., 2000; Malikova et al., 2006b). Current available chemotherapeutic agents have high toxicity towards normal cells in addition to tumor cells. Thus, searching for anti-tumor agents with minimal toxicity towards normal cells is of great value, and plant-derived products are in the great concern. Here we compared the effects of sanguinarine, chelerythrine and chelidonine upon the DNA damage, nucleus integrity and cytotoxicity in primary mouse spleen cells and mouse lymphocytic leukemic cell line, L1210. By comparing the effects of these alkaloids in normal and cancer cells, an in vitro animal model was established which provided important clues for their future applications. 2. Materials and methods 2.1. Extraction and purification of alkaloids
Fig. 1. Structure of the alkaloids.
Sanguinarine, chelerythrine and chelidonine were isolated and purified from roots of the greater celandine by Dr. Maxim Lootsik (see Kaminskyy et al., 2006). The alkaloids were dissolved in 50% ethanol and diluted in culture medium, taking into account that the final concentration of ethanol in culture medium did not exceed 0.1%.
2.2. Cell cultures
chelerythrine-mediated apoptosis occurred in various human tumor cell lines (Chan et al., 2003; Kemeny-Beke et al., 2006; Malikova et al., 2006b; Yamamoto et al., 2001). In addition, chelerythrine exhibited cytotoxic effect against radioresistant and chemoresistant squamous carcinoma cells with delayed tumor growth and mild toxicity in an animal model (Chmura et al., 2000). The usefulness of chelerythrine in combination with photodynamic therapy or ionizing radiation has been implicated (Chmura et al., 1997; Sausville et al., 1998). Sanguinarine induces cell cycle arrest and apoptosis via various pathways (Adhami et al., 2004; Malikova et al., 2006a). The involvement of mitochondrial pathway and Bcl-2 family proteins in apoptosis has been demonstrated (Ahmad et al., 2000). The inhibition of mitogen-activated protein kinase (MAPK) phosphatase, suppression of vascular endothelial growth factor (VEGF)-induced angiogenesis (Eun and Koh, 2004) and down-regulation of adhesion molecules (Tanaka et al., 2001) implicate sanguinarine as a potential agent against tumor progression and metastasis. Sanguinarine induces apoptosis in human squamous carcinoma without significant effect on normal human keratinocytes (Ahmad et al., 2000). A number of studies suggest that sanguinarine is a potential anti-tumor agent (Ding et al., 2002; Kemeny-Beke et al., 2006; Weerasinghe et al., 2001). Chelidonine induces cell cycle arrest and activation of stress-activated protein kinase/Jun kinase (SAK/JNK) pathway. It inhibits tubulin polymerization and disrupts microtubular structure in cells (Panzer et al.,
Mouse lymphocytic leukemic cells, L1210, were obtained from National Collection of Cell Lines at the Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv, Ukraine. Mouse spleen was removed, transferred to Petri dishes, and several small incisions were made on the side of spleen. By squeezing the spleen with phosphate-buffered saline, a single-cell suspension was produced. Yield and viability of cells were assessed by trypan blue exclusion method. Both cell types were cultured in RPMI 1640 medium (Sigma Chem. Co, USA) supplemented with 10% fetal bovine serum (Sigma Chem. Co, USA). Cells were maintained under standard cell culture conditions at 37 C with 5% CO2 in air at 100% humidity.
2.3. Alkaloid treatment Cells were cultured in 96-well plate at 2 105 per well. Chelerythrine, sanguinarine and chelidonine were added to the culture medium at different concentrations (0.5e8 mg/ml), and cells were incubated for 24 h at 37 C.
2.4. Determination of cell viability Cells’ viability was evaluated by trypan blue exclusion method. Cells were stained with 0.1% trypan blue for 10 min at room temperature. The number of live (transparent) and dead (blue) cells was counted in hemocytometric chamber under light microscope. Cell viability was denoted as percentage of live cells. Cell growth was calculated as percentage of increase in amount of cells in 24 h. The 50% lethal dosage (LD50) was determined from doseeresponse curves.
2.5. DNA comet assay at moderate alkaline pH For detection of DNA damage, comet assay at moderate alkaline pH was performed, as described by Kaminskyy et al. (2006). Briefly, cells were
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collected by centrifugation and resuspended in phosphate-buffered saline to final concentration of 3 105 cells/ml. The suspension was mixed with low melting point agarose and rapidly pipetted onto microscopic slides and covered with coverslips. Slides were immersed in lysis buffer (0.5 M EDTA, 2% lauryl-sarcosine, 0.3 mg/ml proteinase K, pH 7.5) for 60 min at 4 C, then for 20 h at 37 C. Slides were equilibrated by careful washing in electrophoresis buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.5) and electrophoresed at 0.6 V/cm for 25 min. The slides were stained with 2 mg/ml ethidium bromide in distilled water. DNA comets were analyzed using fluorescence microscope (LOMO, Russia) with barrier filter of 590 nm under magnification 200 and captured by digital camera. Comet images were analyzed using CometScore software (TriTek Corporation). The tail moment (tail length multiplied by the DNA density in the comet tail) was used as primary measurement for quantification of extent of DNA damage. A minimum of 100 cells were analyzed for each sample and data were transferred into Microsoft Excel software for analysis.
2.6. Morphology assessment of apoptosis Living unfixed cells were stained with fluorescent bis-benzimidazole dye, Hoechst 33342, at a final concentration of 1.5 mM in PBS. Cells were incubated for 20 min at 37 C. Nucleus morphology was analyzed using fluorescence microscope (LOMO, Russia) with 430 nm barrier filter (laser beam excitation at 360 nm) under 400 magnification. Images of random fields were captured by digital camera. Nucleus with chromatin condensation, nuclear fragmentation or apoptotic bodies was identified as apoptotic cells.
2.7. Statistical analysis All experiments were performed in triplicate. The Student’s t-test was used and P < 0.05 were considered as statistical significance. Statistical analyses were performed by Microsoft Excel.
3. Results 3.1. Cell viability The cytotoxic effect of alkaloids on cell growth was determined by trypan blue exclusion method in terms of cell viability (Figs. 2 and 3). Sanguinarine and chelerythrine exhibited cytotoxicity in dose-dependent manner in both cell types. Cells exposed to chelidonine only slightly decreased in viability, but showed complete arrest of cell growth at >4.6 mg/ml (Fig. 3). In primary mouse spleen cells, the LD50 after 24 h with sanguinarine and chelerythrine was 2.8 mg/ml and 4.6 mg/ml, respectively (Fig. 2A). In L1210 cells, the LD50 corresponding values were 1.9 mg/ml and 3.2 mg/ml, respectively (Fig. 2B). L1210 cells showed higher sensitivity to sanguinarine (>2 mg/ml) and chelerythrine (>4 mg/ml) treatment, compared to mouse spleen cells. There were no significant differences in cytotoxity in both cell types treated with chelidonine. 3.2. DNA comet assay In order to estimate DNA damage in cells exposed to alkaloids, comet assay at moderate alkaline pH was performed (Figs. 4 and 5). In both mouse spleen cells and L1210 cells, sanguinarine and chelerythrine showed a dose-dependent increase in the percentage of cells with DNA damage compared to control. Sanguinarine showed a statistically significant higher level of DNA damage at concentration >2 mg/ml in
Fig. 2. Effect of alkaloids on cell viability measured by trypan blue exclusion method in (A) mouse spleen cells and (B) mouse leukemic cells, L1210. Sanguinarine and chelerythrine exhibit cytotoxicity in dose-dependent manner in both mouse spleen cells and L1210 cells. Chelidonine showed only slight decrease in cell viability and did not show significant effect in dose-dependent manner, as compared to control. L1210 cells showed significant slightly higher sensitivity to sanguinarine and chelerythrine treatment, as compared to mouse spleen cells. The means values and standard deviations are shown.
spleen cells and >1 mg/ml in L1210 cells. Under treatment of chelerythrine, intensive DNA damage was seen at >4 mg/ ml in both spleen cells and L1210 cells. Chelidonine did not show any significant effect on DNA damage compared to control. L1210 cells showed significantly higher sensitivity to sanguinarine (4 mg/ml) and chelerythrine (4 mg/ml and 8 mg/ml) treatment compared to mouse spleen cells. In both cell types, sanguinarine at >8 mg/ml showed intensive DNA damage (data not presented). No significant differences were observed in both cell types after treatment of chelidonine. 3.3. Morphology assessment of apoptosis Hoechst 33342 dye penetrates the plasma membrane and stains DNA in the cells. The nuclei of apoptotic cells are
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4. Discussion
Fig. 3. Effect of alkaloids on cell growth in L1210 cells. Cells exposed to chelidonine exhibited complete arrest of cell growth at concentration >4.6 mg/ml.
characterized by chromatin condensation, nuclear fragmentation or presence of apoptotic bodies. We examined morphological changes of cell nuclei in order to assess our findings above (Fig. 6). In L1210 cells, the level of apoptotic features after treatment of sanguinarine and chelerythrine was consistent with cytotoxicity and DNA damage. Chelidonine showed atypical nuclear morphology in L1210 cells, which can be explained by its effect on cell growth and cell cycle arrest in mitosis.
The main goal of our study was to investigate the differences in cytotoxic activity of sanguinarine, chelerythrine and chelidonine in normal and cancer cells. We used primary mouse spleen cells and mouse lymphocytic leukemic cell line, L1210. Spleen cells are mainly composed of lymphocytes, thereby representing the normal counterpart of leukemic cells, while L1210 is mouse leukemia of lymphocytic origin with well characterized features that is commonly used as an in vitro leukemic model. Previous studies showed that chelerythrine and sanguinarine treatment of many cancer cell lines (Adhami et al., 2004; Ahmad et al., 2000; Chmura et al., 2000; KemenyBeke et al., 2006; Malikova et al., 2006b) and several primary cell culture and normal cell lines (Ahmad et al., 2000; Malikova et al., 2006b; Vavreckova et al., 1996a,b) resulted in dose-dependent decrease in cell viability. These studies are consistent with our results, showing that sanguinarine and chelerythrine decrease cell viability significantly in dosedependent manner in both mouse spleen cells and L1210 cells. We found that sanguinarine has significantly higher cytotoxicity than chelerythrine in both cell types, which agrees with a recent report on uveal melanoma cells (Kemeny-Beke et al., 2006). Our study showed that chelidonine only slightly decreased cell viability and did not show significant cytotoxicity as assessed by trypan blue exclusion. Previous studies have shown that cytotoxicity induced by chelidonine is much less
Fig. 4. Effect of alkaloids on DNA damage measured by comet assay in (A) mouse spleen cells þ sanguinarine; (B) L1210 cells þ sanguinarine; (C) mouse spleen cells þ chelerythrine; (D) L1210 cells þ chelerythrine; (E) mouse spleen cells þ chelidonine; and (F) L1210 cells þ chelidonine. Sanguinarine showed statistically significant higher level of DNA damage at concentration >2 mg/ml in spleen cells and >1 mg/ml in L1210 cells. Chelerythrine showed statistically significant higher level of DNA damage at concentration >4 mg/ml in spleen cells and L1210 cells. Chelidonine showed only slightly increase in DNA damage and did not show significant effect in DNA damage, as compared to control. L1210 cells showed significant higher sensitivity to sanguinarine (4 mg/ml) and chelerythrine (4 mg/ml and 8 mg/ml) treatment, as compared to mouse spleen cells (* indicates significant difference comparing to control value at P < 0.05 and þ indicates significant difference of both cell types).
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Fig. 5. Examples of DNA comet images (200). (A) Mouse spleen cells, control; (B) mouse spleen cells þ 4 mg/ml sanguinarine; (C) mouse spleen cells þ 8 mg/ml chelerythrine; (D) mouse spleen cells þ 8 mg/ml chelidonine; (E) L1210 cells, control; (F) L1210 cells þ 4 mg/ml sanguinarine; (G) L1210 cells þ 8 mg/ml chelerythrine; and (H) L1210 cells þ 8 mg/ml chelidonine. No tail detected in (A) and (E). Comets with small or no tail, which are similar to control (D, H). Comets with medium and long tail are showed in (B), (C), (F), and (G). Photos are representative of at least three independent experiments.
effective than by sanguinarine and chelerythrine (KemenyBeke et al., 2006; Vavreckova et al., 1996a,b). This is because the cytotoxic effects of sanguinarine and chelerythrine are attributed to the disruption of plasmatic membrane, which was detected by trypan blue exclusive method. Increased permeability of late apoptotic cells is the consequence of a compromised integrity of the plasma membrane. This corresponds with a previous study, which showed that human keratinocytes are susceptible for sanguinarine and chelerythrine in terms of plasma membrane integrity, resulting in a twofold increase in
the release of lactate dehydrogenase activity (Vavreckova et al., 1996b). On the other hand, chelidonine induced cell growth arrest (Panzer et al., 2001), which is consistent with our results. Comet assay was used to evaluate the level of DNA damage in individual cell. Sanguinarine and chelerythrine exhibited significant increase in DNA damage in dose-dependent manner in both mouse spleen cells and L1210 cells. Sanguinarine rendered higher levels of DNA damage than chelerythrine. Higher tail moments suggested more intensive DNA strand
Fig. 6. Examples of Hoechst 33342-stained cells (400). (A) Mouse spleen cells, control; (B) mouse spleen cells þ 4 mg/ml sanguinarine; (C) mouse spleen cells þ 8 mg/ml chelerythrine; (D) mouse spleen cells þ 8 mg/ml chelidonine; (E) L1210 cells, control; (F) L1210 cells þ 4 mg/ml sanguinarine; (G) L1210 cells þ 8 mg/ml chelerythrine; and (H) L1210 cells þ 8 mg/ml chelidonine. Apoptotic cells with chromatin condensation, nuclear fragmentation, or apoptotic bodies are showed in (B), (C), (F), and (G). Photos are representative of at least three independent experiments.
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breaks. DNA damaged cells possessed typical small head and large tail pattern, called ‘‘hedgehog cells’’, and revealed major DNA fragmentation resulting from late apoptotic events. Chelidonine did not show significant DNA damage in both cell types compared with controls. This observation supports our finding mentioned above, in which both cell types are sensitive to sanguinarine and chelerythrine treatment. Our results are consistent with a previous study in which human gingival fibroblasts and prostate cancer cell lines were used (Malikova et al., 2006b). Nuclear morphology was monitored by Hoechst 33342 staining. Apoptotic morphological features were more prominent in L1210 cells treated with chelerythrine and sanguinarine. Treatment of cells with chelidonine did not show a significant level of apoptosis, while chromatin disorganization occurred in L1210 cells. These findings are consistent with the observations described above. It is known that chelerythrine induced apoptosis in various normal and cancer cell types, such as human gingival fibroblast (Malikova et al., 2006b), squamous cell carcinoma (HNSCC) (Chmura et al., 2000), human leukemia (Chmura et al., 1996; Freemerman et al., 1996), human breast cancer (MCF-7), human colon carcinoma (Chan et al., 2003) and human uveal melanoma (OCM1) (Kemeny-Beke et al., 2006), while sanguinarine induced apoptosis in human erythroleukemia cells (K562) and human prostate carcinoma cells (Adhami et al., 2004). The detail mechanisms of chelerythrine and sanguinarine on apoptosis were extensively studied and well elucidated (Chan et al., 2003; Chaturvedi et al., 1997; Herbert et al., 1990; Hu et al., 2000; Vogt et al., 2005; Yu et al., 2000). Our result showed that L1210 cells are significantly more sensitive to sanguinarine and chelerythrine treatment compared with normal spleen cells in terms of disruption of plasmic membrane integrity and DNA damage. Chelidonine’s effect did not show significant difference between normal spleen cells and L1210 cells. These findings suggested that sanguinarine and chelerythrine are slightly more cytotoxic against cancer cells. Several studies support our findings. At micromolar concentrations, the effects on cell growth inhibition and apoptosis are significantly higher in human squamous carcinoma (A431) cells via induction of apoptosis, in comparison to normal human epidermal keratinocytes (Ahmad et al., 2000). Chelerythrine delays tumor growth in experimental model with relatively mild toxicity to animal (Chmura et al., 2000). In contrast, a recent study demonstrated that chelerythrine and sanguinarine exerted higher toxicity towards normal human gingival fibroblasts in comparison to prostate cancer cell lines (Malikova et al., 2006b). We propose that different cell types exert differential level of sensitivity to alkaloids treatment, although further experiments, both in vivo and in vitro, are crucial for evaluation of these alkaloids and their undesired toxic effect to normal cells. In conclusion, our study demonstrated that (1) in both normal spleen cells and L1210 leukemic cells, the effect of cytotoxicity or disruption of plasma membrane integrity, DNA damage and apoptosis are observed in dose-dependent manner after sanguinarine and chelerythrine treatment; (2) sanguinarine confers
higher level of cytotoxicity and DNA damage than chelerythrine, while chelidonine showed cell growth arrest with minimal cytotoxicity; and (3) L1210 cells were more sensitive to toxic effect of chelerythrine and sanguinarine treatment, when compared to normal mouse spleen cells. These results suggest that chelerythrine and sanguinarine possess slightly selective toxicity against cancer cells. However, further studies are needed to exploit their toxicity towards other normal cell types. The mechanism and signaling pathway of differential toxic effect of these alkaloids towards normal and cancer cells are of great interest for future studies, which are achievable by molecular and genomic approaches. Acknowledgements We thank Dr. Maxim Lootsik (Institute of Cell Biology, National Academy of Sciences, Lviv, Ukraine) for presenting alkaloids and Taras Soroka for his technical assistance in the experiments. References Adhami VM, Aziz MH, Reagan-Shaw SR, Nihal M, Mukhtar H, Ahmad N. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitorecycline cyclin-dependent kinase machinery. Mol Cancer Ther 2004;3:933e40. Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 2000;6:1524e8. Chan SL, Lee MC, Tan KO, Yang LK, Lee AS, Flotow H, et al. Identification of chelerythrine as an inhibitor of BclXL function. J Biol Chem 2003;278:20453e6. Chaturvedi MM, Kumar A, Darnay BG, Chainy GB, Agarwal S, Aggarwal BB. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kappaB activation, IkappaBalpha phosphorylation, and degradation. J Biol Chem 1997;272:30129e34. Chmura SJ, Nodzenski E, Crane MA, Virudachalam S, Hallahan DE, Weichselbaum RR, et al. Cross-talk between ceramide and PKC activity in the control of apoptosis in WEHI-231. Adv Exp Med Biol 1996;406:39e55. Chmura SJ, Mauceri HJ, Advani S, Heimann R, Beckett MA, Nodzenski E, et al. Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation. Cancer Res 1997;57:4340e7. Chmura SJ, Dolan ME, Cha A, Mauceri HJ, Kufe DW, Weichselbaum RR. In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo. Clin Cancer Res 2000;6:737e42. Colombo ML, Bosisio E. Pharmacological activities of Chelidonium majus L. (Papaveraceae). Pharmacol Res 1996;33:127e34. Ding Z, Tang SC, Weerasinghe P, Yang X, Pater A, Liepins A. The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death. Biochem Pharmacol 2002;63:1415e21. Eley BM. Antibacterial agents in the control of supragingival plaque e a review. Br Dent J 1999;186:286e96. Ernst E, Schmidt K. Ukrain e a new cancer cure? A systematic review of randomised clinical trials. BMC Cancer 2005;5:69. Eun JP, Koh GY. Suppression of angiogenesis by the plant alkaloid, sanguinarine. Biochem Biophys Res Commun 2004;317:618e24. Freemerman AJ, Turner AJ, Birrer MJ, Szabo E, Valerie K, Grant S. Role of c-jun in human myeloid leukemia cell apoptosis induced by pharmacological inhibitors of protein kinase C. Mol Pharmacol 1996;49:788e95.
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