Original Papers
Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus glutinosa) and Their Interaction with Anticancer Drugs
Authors
Jelena Dinić 1, Miroslav Novaković 2, Ana Podolski-Renić 1, Sonja Stojković 1, Boris Mandić 3, Vele Tešević 3, Vlatka Vajs 2, Aleksandra Isaković 4, Milica Pešić 1
Affiliations
1 2 3 4
Key words " diarylheptanoid l " Alnus glutinosa l " Betulaceae l " curcumin l " doxorubicin l " cisplatin l " reactive oxygen species l (ROS)
received revised accepted
April 13, 2014 July 3, 2014 July 23, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382993 Published online August 19, 2014 Planta Med 2014; 80: 1088–1096 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Jelena Dinić Institute for Biological Research Department of Neurobiology University of Belgrade Bulevar Despota Stefana 142 11000 Belgrade Serbia Phone: + 38 11 12 07 84 06 Fax: + 38 11 12 76 14 33 [email protected] Correspondence Dr. Milica Pešić Institute for Biological Research Department of Neurobiology University of Belgrade Bulevar Despota Stefana 142 11000 Belgrade Serbia Phone: + 38 11 12 07 84 06 Fax: + 38 11 12 76 14 33 [email protected]
Institute for Biological Research, Department of Neurobiology, University of Belgrade, Belgrade, Serbia Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, Serbia Faculty of Chemistry, University of Belgrade, Belgrade, Serbia Faculty of Medicine, University of Belgrade, Belgrade, Serbia
Abstract !
Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI–H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the
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messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.
Abbreviations !
AV: CI: CPT: CUR: DHE: DOX: gapdh:
annexin-V-FITC combination index cisplatin curcumin dihydroethidium doxorubicin glyceraldehyde 3-phosphate dehydrogenase HIF-1α: hypoxia-inducible factor-1α MFI: mean fluorescence intensity Mn-SOD: manganese superoxide dismutase NSCLC: non-small cell lung carcinoma PBMC: peripheral blood mononuclear cells PI: propidium iodide Rho 123: rhodamine 123 ROS: reactive oxygen species RT‑PCR: reverse transcription polymerase chain reaction Supporting information available online at http://www.thieme-connect.de/products
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Introduction Diarylheptanoids are phenolic compounds with a heptane chain between two aromatic rings [1]. They are abundant in Alnus, Zingiber, Curcuma, Alpinia, Betula, and Myrica genera. One of the most investigated diarylheptanoids is CUR, a turmeric component used in traditional Asian medicine for a variety of disorders. CUR was first isolated in 1815 from the rhizome of Curcuma longa (Zingiberaceae) and was shown to possess a wide range of biological activities and potential therapeutic effects against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune, and neoplastic diseases [2]. Other diarylheptanoids are increasingly recognized as potential therapeutic agents with antioxidative [3], anti-inflammatory [4], cytotoxic, anticancer [5], hepatoprotective [6], and neuroprotective activities [7]. Diarylheptanoids can also enhance the efficiency of conventional anticancer drugs creating a synergistic effect, which leads to cancer cell apoptosis [8]. Besides promoting cell death, alone and in combination with conventional chemotherapeutics, diarylheptanoids also regulate cellular antistress responses. CUR has been shown to modulate the activity of transcription factors that regulate the expression of genes involved in free radical scavenging such as catalase, MnSOD, heme oxygenase-1, and HIF-1α [9–11]. Increased expression of MnSOD and HIF-1α has been linked to the induction of drug resistance and promotion of cell survival [12–14]. Limited focus was put on investigating diarylheptanoids isolated from Alnus glutinosa L. (Betulaceae), especially from its bark [15]. For that purpose, we evaluated the anticancer and chemoprotective effects of two diarylheptanoids: platyphylloside 5(S)-1,7-di (4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) [16] and its analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated " Fig. 1) [17]. Our previous results from the bark of black alder (l showed a DNA protective effect of diarylheptanoids from A. glutinosa bark in normal human lymphocytes [18] as well as considerable activity against cancer cells [17]. We have assessed the effect of the abovementioned diarylheptanoids in combination with CPT or DOX. CPT is an alkylating agent whose specific mechanism of action is not entirely understood; however, it is known to cause cross-linking and adduct formation by intercalating with DNA strands and possess free radical generating properties [19]. In addition to the formation of free radicals, DOX, an anthracycline drug, also intercalates DNA and causes inhibition of topoisomerase II [20]. Therefore, our main aim in evaluating these diarylheptanoids therapeutic benefit was to study their effects when combined with classic chemotherapeutics in both cancer and normal cells.
Results !
The effects of 1, 2, and CUR as well as classic chemotherapeutics DOX and CPT on cell growth were analyzed after 72 h by the MTT assay. The IC50 values of single and combined treatments are " Fig. 2 and Table 1S. The IC summarized in l 50s for DOX obtained in NCI-H460 and HaCaT cells were 28 nM and 7 nM, respectively, suggesting that DOX is not selective towards cancer cells in this experimental system. CPT exerted a similar inhibitory effect on both cell lines with IC50 values of 1.1 µM in NCI-H460 and 1.8 µM in HaCaT cells. Among the diarylheptanoids (1, 2, and CUR), only 2 was 1.6 times more efficient in cancer cells when
Fig. 1 Chemical structure of diarylheptanoids from A. glutinosa: platyphylloside (1) and its analog with a p-coumaroyl group (2).
compared with HaCaT. Furthermore, we have evaluated the effects of all compounds after continuous 48 h treatment in nonstimulated normal PBMCs from healthy donors. The IC50 values for all compounds obtained in the primary culture were consid" Fig. 2). erably higher in comparison to cancer cells (l To investigate the interaction of diarylheptanoids with classic anticancer drugs, we combined them with DOX and CPT. After 6 h pretreatment with 10 µM of each diarylheptanoid, NCI-H460 and HaCaT cells were incubated for an additional 72 h with five different concentrations of DOX or CPT (described in Materials and Methods). The incubation for the PBMC culture lasted 48 h. Compounds 1, 2, and CUR decreased the sensitivity of cancer and normal cell lines as well as PBMC to classic chemotherapeutics, which is illustrated with increases in IC50 concentrations for " Fig. 2). DOX and CPT in combined treatments (l These results were subjected to CalcuSyn software, which analyzes the nature of the interaction between two different drugs (synergism/antagonism). The majority of the combinations used in this course of treatment demonstrated a pronounced antago" Fig. 2). Moreover, differences between sinnistic (CI > 1) effect (l gle and combined treatments were more prominent in normal cells. To investigate the potential of diarylheptanoids to induce cell death, NCI-H460 and HaCaT cells treated with diarylheptanoids alone or in combination with DOX were subjected to AV/PI stain" Fig. 3. NCI-H460 cells were ing. The results are summarized in l treated with 10 µM, 25 µM, and 50 µM diarylheptanoids for 72 h " Fig. 3 A). The percentage of apoptotic and necrotic cells gradu(l " Fig. 3 A). ally increased in comparison with untreated control (l Pretreatment with 10 µM of 1 and 2 did not considerably change " Fig. 3 B). the cell death induction observed with 20 nM DOX (l Compounds 1 and 2 in combination with DOX increased the percent of apoptotic and necrotic cells when compared with DOX " Fig. 3 B). Contraalone (from 15 % to 23% and 25%, respectively; l Dinić J et al. Antioxidative Activity of …
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Fig. 2 Diarylheptanoids increase IC50 values and antagonize the effect of doxorubicin and cisplatin in NCI-H460 and HaCaT cell lines and peripheral blood mononuclear cells. IC50 values for 1, 2, and CUR (A). IC50 values for DOX after pretreatment with 1, 2, and CUR (B). IC50 values for CPT after pretreatment with 1, 2, and CUR (C). Most combination treatments exhibited an antagonistic effect (CI > 1). Exception where CI < 1 is labeled with #. Cell
rily, 10 µM CUR significantly improved the DOX effect by inducing " Fig. 3 B). In HaCaT cells, 1 cell death in more than 50 % of cells (l (5 µM) and DOX (40 nM), alone, induced a similar effect, while 2 (5 µM) did not significantly change the percentage of dead cells in " Fig. 3 C). Both diarycomparison with the untreated control (l lheptanoids reverted the effect of DOX in combination, confirm" Fig. 3 C). However, ing the previously observed antagonism (l the antagonism was more pronounced when 2 was combined " Fig. 3 C). with DOX (l To visualize the localization of DOX after diarlyheptanoid treatment (5 µM for 72 h), NCI-H460 and HaCaT cells were exposed
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growth after a single DOX treatment and after the combination treatment with 1, 2, and CUR (D, F). Cell growth after a single CPT treatment and after the combination treatment with 1, 2, and CUR (E, G). Where not indicated, the Y-axis represents the percentage of cell growth compared to untreated cells. Statistical significance between single and combined treatments is presented as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
to 10 µM DOX for 2 h. In order to achieve microscopically detectable levels of fluorescence, DOX was applied at concentrations significantly higher than the IC50 values. Diarylheptanoid 1 caused partial accumulation of DOX in the cytoplasm of NCI" Fig. 4 A). The extranuclear localization H460 and HaCaT cells (l of DOX observed in NCI-H460 cells was less pronounced " Fig. 4 A). (l To test the effects of diarylheptanoids on mitochondrial structure, NCI-H460 and HaCaT cells were treated with 5 µM of diarylheptanoids for 72 h. Then, a high concentration of DOX (20 µM) was added to rapidly disrupt the mitochondria. After 2 h of DOX
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Fig. 3 Cell death induction by diarylheptanoids in cancer and normal cells. Analysis performed on untreated and NCI-H460 cells treated with increasing concentrations of 1, 2, and CUR for 72 h (A), 20 nM DOX and combinations of 10 µM of 1, 2, and CUR with 20 nM of DOX (B), untreated and HaCaT cells treated with 5 µM of 1 and 2 and 40 nM of DOX for 72 h, and combinations of 5 µM of 1 and 2 with 40 nM of DOX (C). The samples were analyzed for green fluorescence (AV, FL1-H) and red fluorescence (PI, FL2-H) by flow cytometry. (Color figure available online only.)
treatment, cells were labeled with Rho123 and imaged live " Fig. 4 B). All diarylheptanoids exhibited a protective effect on (l the mitochondrial structure. However, 1 had the most prominent effect in both cell lines. This compound preserved the tubular structure of mitochondria similar to the untreated control de" Fig. 4 B). spite subsequent DOX application (l To test superoxide anion production, NCI-H460 and HaCaT cells were treated with 5 µM of diarylheptanoids for 72 h. To assess the effect of the combinations, the cells were pretreated for 6 h with diarylheptanoids, and subsequently incubated with 2 µM CPT for an additional 72 h. Superoxide anion generation was detected by DHE staining and assessed by flow cytometric analysis " Fig. 5). The intensity of DHE in and fluorescence microscopy (l the combined treatments significantly declined in both cell lines in comparison with CPT alone. The decrease in superoxide anion production was more pronounced in HaCaT cells, with diarylhep-
" Fig. 5 A). These effects tanoid 2 exhibiting the strongest effect (l " Fig. 5 B). were confirmed by DHE imaging (l To study the mechanism behind the protective effect of diarylheptanoids observed in cells exposed to the damaging agents DOX and CPT, we analyzed the influence of diarylheptanoids on mRNA expression of enzymes with significant roles in oxidative stress, MnSOD and HIF-1α. The expression of both enzymes was analyzed in the RNA samples isolated from NCI-H460 cells, un" Fig. 6). All diaryltreated and treated with 10 µM 1, 2, and CUR (l heptanoids significantly increased the level of MnSOD mRNA ex" Fig. 6 A). Similarly, pression compared to the untreated control (l compounds 1 and 2 caused a significant increase in HIF-1α mRNA expression, while the increase observed with CUR was not statis" Fig. 6 B). tically significant (l
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Fig. 4 Diarylheptanoid 1 causes doxorubicin retention in the cytoplasm and reduces mitochondrial fragmentation induced by doxorubicin in NCI-H460 and HaCaT cells. DOX retention in the cytoplasm after diarylheptanoid 1 application in NCI-H460 and HaCaT cells (A). Subcellular localization of DOX was visualized after a 1-h incubation with 10 µM of DOX at 37 °C. Arrows point to extranuclear DOX. Nuclei were counterstained with Hoechst 33 342. Scale bar = 100 µm. Diarylheptanoid 1 reduces mitochondrial fragmentation induced by DOX in NCIH460 and HaCaT cells (B). Untreated cells possess a characteristic tubular mitochondrial structure. Mitochondrial fragmentation was induced by 20 µM of DOX for 2 h. Cells pretreated with 5 µM of 1 for 72 h were protected from mitochondrial fragmentation. Mitochondria were labeled with Rho123. Arrows point to the mitochondria which retained the tubular structure after DOX treatment. Nuclei were counterstained with Hoechst 33 342. Scale bar = 100 µm. (Color figure available online only.)
Fig. 5 Diarylheptanoid 2 decreases superoxide anion levels generated by cisplatin in NCI-H460 and HaCaT cells. Flow cytometric profiles of untreated and treated NCI-H460 and HaCaT cells evaluated after labeling with DHE (A). NCI-H460 and HaCaT cells labeled with DHE were visualized on a fluorescent microscope (B). Scale bar = 200 µm. (Color figure available online only.)
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Fig. 6 Diarylheptanoids 1 and 2 increase the level of MnSOD and HIF-1α mRNA expression in NCI-H460 cells. The effect of 10 µM of 1, 2, and CUR on MnSOD mRNA expression in NCI-H460 cells (A). The PCR products were separated on agarose gel next to a 100 bp DNA ladder. The expression of the MnSOD gene was calculated relative to β-actin expression. Statistical significance between treated and untreated (control) cells is presented as p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*). The effect of 10 µM of 1, 2, and CUR on HIF-1α mRNA expression in NCI-H460 cells (B). The PCR products were separated on agarose gel next to a 100 bp DNA ladder. The expression of the HIF-1α gene was calculated relative to gapdh expression. Statistical significance between treated and untreated (control) cells is presented as p < 0.05 (*).
Discussion !
Researchers who explore bioactive substances from plants have made increasing efforts to find safer and more efficient anticancer drugs. Particularly, plants known from folk medicine are a valuable source of new anticancer agents [21]. However, some natural sources, not considered previously, were shown to be plentiful in new bioactive substances [22]. Recently, we have explored the anticancer activity of 24 diarylheptanoids from the bark of A. glutinosa. Ten of them have not been reported previously [17]. Herein, we investigated the effects of a known platyphylloside (1) and its newly discovered analog with a p-coumaroyl group (2). Both compounds considerably inhibited the growth of chemosensitive and chemoresistant NSCLC in vitro with an IC50 < 30 µM, showing the best effect among the tested compounds [17]. Importantly, we found that diarylheptanoid 2 was less efficient in HaCaT cells, while the efficacy of both 1 and 2 was reduced in PBMCs. This implies that when applied alone, diarylheptanoids are more active in cancer cells than in normal cells. The selectivity towards cancer cells is a prerequest in choosing good drug candidates for cancer treatment. In the diarylheptanoid classic chemotherapeutics interaction study, our results demonstrated a suppressive effect of 1 and 2 on either DOX or CPT activity. Although present in cancer cells, the observed antagonism was more pronounced in normal cells. Because of this antagonism, IC50 values for DOX and CPT were sig-
nificantly raised in combination with 1 and 2. A similar effect was achieved with CUR, but it was less apparent in comparison with 1 and 2. Although our previous results suggested that diarylheptanoids could be valuable for cancer treatment, it has to be noticed that in combination with either DOX or CPT, diarylheptanoids could diminish the benefit of these drugs. On the other hand, diraylheptanoids may protect normal cells from damage caused by classic chemotherapeutics. Further, we discuss whether diarylheptanoids could be advantageous for treatment in cancer patients. The induction of apoptosis has been implicated as an important mechanism underlying cytotoxic activities of many chemotherapeutic agents. In our present study, we investigated cell death induction by diarylheptanoids in the NSCLC cell line NCI-H460. We confirmed by annexin-V/PI staining that 1, 2, and CUR significantly increase the percent of apoptotic cancer cells in a concentration-dependent manner. These results provided clear evidence of the apoptotic potential of diarylheptanoids in cancer cells. However, 1 and 2 failed to improve the DOX effect, while CUR considerably enhanced cell death induction in combination with DOX. Both diarylheptanoids diminished the effect of DOX in normal cells. Particularly, 2 decreased the cell death induced with DOX by reverting the effect to the level observed in untreated control. Considering that diarylheptanoids did not affect DOX-induced cell death in cancer cells but only failed to improve it, these compounds could be beneficial for the protection of normal cells in cancer patients. DOX accumulates in the cell nucleus intercalating into DNA inhibiting macromolecular biosynthesis [23]. The main effect is on the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription [20]. DOX also induces histone eviction from chromatin affecting the regulation of the DNA damage response in DOX-exposed cells [24]. We have observed the retention of DOX in the cytoplasm of both NCI-H460 and HaCaT cells pretreated with diarylheptanoid 1. Considering that DOX needs to enter the nucleus to reach its target molecules DNA and topoisomerase II, this finding may present one of the mechanisms behind the antagonistic interaction between 1 and DOX. However, this could be only one of the reasons for the observed antagonism of diarylheptanoids with DOX. Some authors showed that CUR might decrease topoisomerase II expression or act as topisomerase II poison [25–26], while CUR-induced topoisomerase-DNA complexes might be mediated by ROS [27]. If the diarylheptanoids that we investigated resemble the pattern of the CUR mechanism, they may antagonize the DOX effect by reducing its target availability. Although it is generally accepted that the toxicity of anticancer drugs is the consequence of their capacity to cause genomic DNA damage, they also exert important effects in other cellular compartments such as mitochondria [28]. One of the mechanisms proposed to be responsible for the toxicity of DOX is the generation of ROS, which mostly affects the mitochondria function [29]. One µM of DOX was enough to disrupt the tubular structure of mitochondria in H9c2 myoblast cells after 24 h treatment [30]. We have shown that DOX treatment increased mitochondrial fragmentation in NCI-H460 and HaCaT cells, which was illustrated by Rho 123 staining. When cells were pretreated with 1, the DOX effect was attenuated, leading to the mitochondrial structure recovering. Particularly under these conditions, the mitochondria structure resembles the structure observed in untreated cells. Other authors showed that antioxidants suppress
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the mitochondrial transmembrane potential decrease associated with the DNA damaging agentʼs action [31]. Therefore, we assumed that diarylheptanoids could scavenge ROS and act as antioxidants antagonizing DOX and CPT effects. It is known that ROS plays an important role in CPT cytotoxicity [19]. We compared the effect of 2 µM CPT alone and in combination with 10 µM of 2 on superoxide anion production in NCIH460 and HaCaT cells. CPT in this concentration exerted significant inhibition of cell growth in both the cancer and non-transformed cell lines. Consequently, we observed the increase at the superoxide anion level after CPT application. In cells pretreated with 2, superoxide anion production was efficiently decreased, suggesting that the diarylheptanoids capacity to antagonize CPT cytotoxicity is due to its ROS scavenging ability. Importantly, the reversal effect of 2 on ROS levels was irrelevant in cancer cells and pronounced in normal human keratinocytes, suggesting that the chemoprotection effect is only in normal cells. Hypoxia or pretreatment with antioxidant agents may attenuate death induction by DNA damaging drugs [32]. Therefore, we have examined the effects of 1 and 2 on Mn-SOD and HIF-1α mRNA levels in cancer cells. Superoxide dismutases are enzymes which catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide, and represent an important antioxidant defense in cells exposed to high levels of oxygen [33]. Mn-SOD is present in mitochondria and peroxisomes. HIF-1, controlled by the rapid stabilization of the HIF-1α subunit, is an essential transcriptional factor in the cellular and systemic responses to hypoxia [34]. Both enzymes have an impact on the intracellular oxidative status leading to the efficient elimination of ROS. We found that 1 and 2 significantly increased the expression of Mn-SOD and HIF-1α mRNA. In comparison with CUR, the effect of 1 and 2 was more apparent. These results suggest that diarylheptanoids could be considered anticancer agents with antioxidant properties. Treatment with a DNA topoisomerase inhibitor, such as DOX, and with an alkylating agent, such as CPT, may cause ROS accumulation in cancer and non-transformed normal cells. These effects could be suppressed by the cytoprotective action of diarylheptanoids found in the present investigation. These compounds may contribute to the protection of normal cells, which are sensitive to the formation of free radicals in the course of chemotherapy. However, their use cannot improve the efficacy of anticancer drugs. Further studies are necessary, in this respect, to confirm diarylheptanoids as candidates for treatment in cancer patients.
Chemicals RPMI 1640 medium, DMEM medium, penicillin–streptomycin solution, antibiotic–antimycotic solution, L-glutamine, and trypsin/EDTA were purchased from PAA. FBS, Rho123, and DMSO were obtained from Sigma-Aldrich Chemie GmbH. PI and AV were purchased from Abcam. DHE was obtained from Molecular Probes®, Invitrogen.
Cell culture The NCI-H460 cell line was purchased from the American Type Culture Collection. NCI-H460 cells were maintained in RPMI 1640 supplemented with 10 % FBS, 2 mM L-glutamine, 4.5 g/L glucose, 10 000 U/mL penicillin, 10 mg/mL streptomycin, and 25 mg/mL amphotericin B solution at 37 °C in a humidified 5 % CO2 atmosphere. The HaCaT cell line (normal human keratinocytes obtained from CLS – Cell Lines Service) was a generous gift from Prof. Andra Jorg, Division of Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany. HaCaT cells were cultured in DMEM supplemented with 10% FBS, 4 g/L glucose, L-glutamine (2 mM), 5000 U/ml penicilin, and 5 mg/mL streptomycin solution.
Isolation of peripheral blood mononuclear cells Heparinized blood taken from healthy volunteers and PBMCs were isolated by centrifugation in lymphocyte separation medium (Ficoll, ICN Biomedicals), according to the manufacturerʼs instructions.
Cytotoxicity by the MTT assay Cell viability was assessed by the MTT assay (Sigma). Briefly, cells were seeded in 96-well microplates and treated with 1, 2, DOX, and CPT for 72 h. In combined treatments, cells were pretreated with 5, 10, and 25 µM of 1, 2, or CUR at 37 °C for 6 h and subsequently treated with increasing concentrations of DOX or CPT. The absorbance of formazan dye was measured at 540 nm using an automatic microplate reader (LKB 5060–006 Micro Plate Reader). Growth inhibition (I) was determined according to the following equation: I (%) = [1 − (Atreated sample/Auntreated control)] × 100 where A is absorbance. The IC50 value was defined as the concentration of each drug that inhibited cell growth by 50%. The IC50 was calculated by linear regression analysis using Excel software.
Materials and Methods !
Median effect analysis
Drugs
The nature of the interaction between different drugs was analyzed using CalcuSyn software (Biosoft) that uses the CI method [35], based on the multiple drug effect equation. At least three data points were used for each single drug in each designed experiment. The non-constant ratio combination was chosen to assess the effect of both drugs in combination. The results are presented by a fraction-affected CI graph. Values of CI < 1 point to a pronounced additive effect or synergism, i.e., the smaller the value, the greater the degree of synergy. A value of CI = 1 indicates an additive effect, and values of CI > 1 point to an antagonistic effect.
DOX solution was obtained from Ebewe Arzneimittel GmbH. CPT was obtained from Pfizer (Perth) Pty Ltd. Both drugs were diluted in sterile water as was prescribed for human use. CUR was purchased from Sigma-Aldrich Chemie GmbH and diluted in absolute ethanol. According to the manufacturer, the purity of CUR was ≥ 98.0 % (HPLC). Diarylheptanoids 1 and 2 were isolated from A. glutinosa bark, as described previously [17]. The purity of tested compounds 1 and 2 was higher than 98 % as determined by HPLC‑DAD (280 nm) and 1H NMR [17, 18].
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Cell death detection The percentages of apoptotic, necrotic, and viable cells were determined by AV and PI labeling. NCI-H460 cells were incubated overnight in 6-well plates and subjected to single treatments with 10, 25, and 50 µM of 1, 2, and CUR. In combined treatments, NCI-H460 cells were pretreated with 10 µM of 1, 2, or CUR, while HaCaT cells were pretreated with 5 µM of 1 or 2 at 37 °C for 6 h and subsequently treated with 20 nM or 40 nM DOX, respectively. After 72 h, the attached and floating cells were collected by centrifugation. AV/PI staining was performed according to the manufacturerʼs instructions and cells were analyzed within 1 h by flow cytometry. The fluorescence intensity (green FL1-H and red FL2-H) was measured on a FACSClibur flow cytometer (Becton Dickinson). In each sample, 10 000 cells were recorded (gated to exclude cell debris), and the percentages of viable (AV- PI-), early apoptotic (AV+ PI−), apoptotic and necrotic (AV+ PI+), and already dead (AV− PI+) cells were analyzed by CellQuest Pro data analysis software.
Superoxide anion detection Flow cytometric analysis of DHE fluorescence intensity was used to detect the superoxide anion level in cells. NCI-H460 and HaCaT cells were incubated overnight in 6-well plates, then pretreated with 5 µM of 1, 2, and CUR at 37 °C for 6 h and subsequently treated with 2 µM CPT. After 72 h, adherent cells were harvested by trypsinization and incubated in adequate medium with 1 µM DHE for 30 min at 37 °C in the dark. Cells were subsequently washed twice in PBS, and DHE fluorescence was analyzed by flow cytometry (excitation 488 nm and emission 585 nm, FL2-H channel). MFI was calculated after correction for autofluorescence.
on a spectrophotometer, and quality was determined by agarose gel electrophoresis. RT reactions using 2 µg of total RNA were performed with a high-capacity cDNA reverse transcription kit (Applied Biosystems), following the manufacturerʼs instructions. PCR reactions were performed with primers specific for MnSOD and HIF-1α [36–37]. β-actin [38] and gapdh [39] were used as internal controls and coamplified with MnSOD and HIF-1α, respectively. The PCR reactions for MnSOD were performed on a GeneAmp® PCR System 9700 (Applied Biosystems) under the following conditions: initial denaturizing at 94 °C for 5 min, 27 cycles at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, and at 4 °C indefinitely. For the amplification of HIF-1α, 25 cycles were performed and the annealing temperature was decreased to 56 °C. The gapdh primers were used at a 1 : 5 ratio to HIF-1α primers in order to attain linear amplification conditions. The β-actin primers were used at a 1 : 1 ratio to MnSOD primers in order to attain linear amplification conditions. The PCR products were separated in 2 % agarose gels stained with ethidium bromide. MultiAnalyst/PC Software Image Analysis (Bio-Rad Gel Doc 1000) was employed for densitometry analysis.
Statistical analysis Statistical analysis was performed by Statistica 6.0 software. The differences between groups were examined by the Studentʼs ttest. Statistical significance was accepted if p < 0.05.
Supporting information The IC50 values of compounds 1, 2, and CUR as well as classic chemotherapeutics DOX and CPT, in single and combined treatments with diarylheptanoids, in NCI-H460 and HaCaT cells and PBMCs are available as Supporting Information.
Fluorescence microscopy NCI-H460 and HaCaT cells were seeded in 8-chamber slides (Nunc) or glass bottom petri dishes and allowed to grow at 37 °C overnight before treatment. To detect superoxide anion, cells were washed in PBS and labeled with 1 µM DHE, a superoxide indicator dye, for 30 min at 37 °C. Live NCI-H460 cells were examined under a Zeiss Axiovert fluorescent microscope (Carl Zeiss Foundation) using AxioVision 4.8 software. HaCaT cells were analyzed under a Leica fluorescent microscope (Leica Microsystems DMIL) using Leica Application Suite software (version 2.8.1). To evaluate changes in the mitochondrial structure, cells were washed in PBS and labeled for 30 min at 37 °C with 5 µM of Rho123, which accumulates in the mitochondria of living cells. Nuclei were counterstained with Hoechst 33 342 (Sigma-Aldrich Chemie) for 15 min at room temperature, and live cells were examined under a Zeiss Axiovert fluorescent microscope. To examine subcellular localization of DOX, cells were incubated with 10 µM of DOX for 1 h at 37 °C. After washing in PBS, nuclei were counterstained with Hoechst 33 342 for 15 min at room temperature and cells were fixed with 4 % paraformaldehyde. Subcellular localization of DOX, which possesses inherent red fluorescence, was analyzed under a Zeiss Axiovert fluorescent microscope.
RNA extraction and reverse transcription polymerase chain reaction Total RNA was extracted from untreated NCI-H460 cells and cells treated with 10 µM of 1, 2, and CUR at 37 °C for 72 h. The isolation was carried out using Trizol (Invitrogen Life Technologies), according to the manufacturerʼs instructions. RNA was quantified
Acknowledgements !
This research was supported by the Ministry of Education, Science and Technological Development of Serbia (grant nos. III 41031 and III 41025).
Conflict of Interest !
The authors declare no conflict of interest
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Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus glutinosa) and Their Interaction with Anticancer Drugs
Authors
Jelena Dinić 1, Miroslav Novaković 2, Ana Podolski-Renić 1, Sonja Stojković 1, Boris Mandić 3, Vele Tešević 3, Vlatka Vajs 2, Aleksandra Isaković 4, Milica Pešić 1
Affiliations
1 2 3 4
Key words " diarylheptanoid l " Alnus glutinosa l " Betulaceae l " curcumin l " doxorubicin l " cisplatin l " reactive oxygen species l (ROS)
received revised accepted
April 13, 2014 July 3, 2014 July 23, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382993 Published online August 19, 2014 Planta Med 2014; 80: 1088–1096 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Jelena Dinić Institute for Biological Research Department of Neurobiology University of Belgrade Bulevar Despota Stefana 142 11000 Belgrade Serbia Phone: + 38 11 12 07 84 06 Fax: + 38 11 12 76 14 33 [email protected] Correspondence Dr. Milica Pešić Institute for Biological Research Department of Neurobiology University of Belgrade Bulevar Despota Stefana 142 11000 Belgrade Serbia Phone: + 38 11 12 07 84 06 Fax: + 38 11 12 76 14 33 [email protected]
Institute for Biological Research, Department of Neurobiology, University of Belgrade, Belgrade, Serbia Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, Serbia Faculty of Chemistry, University of Belgrade, Belgrade, Serbia Faculty of Medicine, University of Belgrade, Belgrade, Serbia
Abstract !
Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI–H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the
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messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.
Abbreviations !
AV: CI: CPT: CUR: DHE: DOX: gapdh:
annexin-V-FITC combination index cisplatin curcumin dihydroethidium doxorubicin glyceraldehyde 3-phosphate dehydrogenase HIF-1α: hypoxia-inducible factor-1α MFI: mean fluorescence intensity Mn-SOD: manganese superoxide dismutase NSCLC: non-small cell lung carcinoma PBMC: peripheral blood mononuclear cells PI: propidium iodide Rho 123: rhodamine 123 ROS: reactive oxygen species RT‑PCR: reverse transcription polymerase chain reaction Supporting information available online at http://www.thieme-connect.de/products
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Introduction Diarylheptanoids are phenolic compounds with a heptane chain between two aromatic rings [1]. They are abundant in Alnus, Zingiber, Curcuma, Alpinia, Betula, and Myrica genera. One of the most investigated diarylheptanoids is CUR, a turmeric component used in traditional Asian medicine for a variety of disorders. CUR was first isolated in 1815 from the rhizome of Curcuma longa (Zingiberaceae) and was shown to possess a wide range of biological activities and potential therapeutic effects against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune, and neoplastic diseases [2]. Other diarylheptanoids are increasingly recognized as potential therapeutic agents with antioxidative [3], anti-inflammatory [4], cytotoxic, anticancer [5], hepatoprotective [6], and neuroprotective activities [7]. Diarylheptanoids can also enhance the efficiency of conventional anticancer drugs creating a synergistic effect, which leads to cancer cell apoptosis [8]. Besides promoting cell death, alone and in combination with conventional chemotherapeutics, diarylheptanoids also regulate cellular antistress responses. CUR has been shown to modulate the activity of transcription factors that regulate the expression of genes involved in free radical scavenging such as catalase, MnSOD, heme oxygenase-1, and HIF-1α [9–11]. Increased expression of MnSOD and HIF-1α has been linked to the induction of drug resistance and promotion of cell survival [12–14]. Limited focus was put on investigating diarylheptanoids isolated from Alnus glutinosa L. (Betulaceae), especially from its bark [15]. For that purpose, we evaluated the anticancer and chemoprotective effects of two diarylheptanoids: platyphylloside 5(S)-1,7-di (4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) [16] and its analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated " Fig. 1) [17]. Our previous results from the bark of black alder (l showed a DNA protective effect of diarylheptanoids from A. glutinosa bark in normal human lymphocytes [18] as well as considerable activity against cancer cells [17]. We have assessed the effect of the abovementioned diarylheptanoids in combination with CPT or DOX. CPT is an alkylating agent whose specific mechanism of action is not entirely understood; however, it is known to cause cross-linking and adduct formation by intercalating with DNA strands and possess free radical generating properties [19]. In addition to the formation of free radicals, DOX, an anthracycline drug, also intercalates DNA and causes inhibition of topoisomerase II [20]. Therefore, our main aim in evaluating these diarylheptanoids therapeutic benefit was to study their effects when combined with classic chemotherapeutics in both cancer and normal cells.
Results !
The effects of 1, 2, and CUR as well as classic chemotherapeutics DOX and CPT on cell growth were analyzed after 72 h by the MTT assay. The IC50 values of single and combined treatments are " Fig. 2 and Table 1S. The IC summarized in l 50s for DOX obtained in NCI-H460 and HaCaT cells were 28 nM and 7 nM, respectively, suggesting that DOX is not selective towards cancer cells in this experimental system. CPT exerted a similar inhibitory effect on both cell lines with IC50 values of 1.1 µM in NCI-H460 and 1.8 µM in HaCaT cells. Among the diarylheptanoids (1, 2, and CUR), only 2 was 1.6 times more efficient in cancer cells when
Fig. 1 Chemical structure of diarylheptanoids from A. glutinosa: platyphylloside (1) and its analog with a p-coumaroyl group (2).
compared with HaCaT. Furthermore, we have evaluated the effects of all compounds after continuous 48 h treatment in nonstimulated normal PBMCs from healthy donors. The IC50 values for all compounds obtained in the primary culture were consid" Fig. 2). erably higher in comparison to cancer cells (l To investigate the interaction of diarylheptanoids with classic anticancer drugs, we combined them with DOX and CPT. After 6 h pretreatment with 10 µM of each diarylheptanoid, NCI-H460 and HaCaT cells were incubated for an additional 72 h with five different concentrations of DOX or CPT (described in Materials and Methods). The incubation for the PBMC culture lasted 48 h. Compounds 1, 2, and CUR decreased the sensitivity of cancer and normal cell lines as well as PBMC to classic chemotherapeutics, which is illustrated with increases in IC50 concentrations for " Fig. 2). DOX and CPT in combined treatments (l These results were subjected to CalcuSyn software, which analyzes the nature of the interaction between two different drugs (synergism/antagonism). The majority of the combinations used in this course of treatment demonstrated a pronounced antago" Fig. 2). Moreover, differences between sinnistic (CI > 1) effect (l gle and combined treatments were more prominent in normal cells. To investigate the potential of diarylheptanoids to induce cell death, NCI-H460 and HaCaT cells treated with diarylheptanoids alone or in combination with DOX were subjected to AV/PI stain" Fig. 3. NCI-H460 cells were ing. The results are summarized in l treated with 10 µM, 25 µM, and 50 µM diarylheptanoids for 72 h " Fig. 3 A). The percentage of apoptotic and necrotic cells gradu(l " Fig. 3 A). ally increased in comparison with untreated control (l Pretreatment with 10 µM of 1 and 2 did not considerably change " Fig. 3 B). the cell death induction observed with 20 nM DOX (l Compounds 1 and 2 in combination with DOX increased the percent of apoptotic and necrotic cells when compared with DOX " Fig. 3 B). Contraalone (from 15 % to 23% and 25%, respectively; l Dinić J et al. Antioxidative Activity of …
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Fig. 2 Diarylheptanoids increase IC50 values and antagonize the effect of doxorubicin and cisplatin in NCI-H460 and HaCaT cell lines and peripheral blood mononuclear cells. IC50 values for 1, 2, and CUR (A). IC50 values for DOX after pretreatment with 1, 2, and CUR (B). IC50 values for CPT after pretreatment with 1, 2, and CUR (C). Most combination treatments exhibited an antagonistic effect (CI > 1). Exception where CI < 1 is labeled with #. Cell
rily, 10 µM CUR significantly improved the DOX effect by inducing " Fig. 3 B). In HaCaT cells, 1 cell death in more than 50 % of cells (l (5 µM) and DOX (40 nM), alone, induced a similar effect, while 2 (5 µM) did not significantly change the percentage of dead cells in " Fig. 3 C). Both diarycomparison with the untreated control (l lheptanoids reverted the effect of DOX in combination, confirm" Fig. 3 C). However, ing the previously observed antagonism (l the antagonism was more pronounced when 2 was combined " Fig. 3 C). with DOX (l To visualize the localization of DOX after diarlyheptanoid treatment (5 µM for 72 h), NCI-H460 and HaCaT cells were exposed
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growth after a single DOX treatment and after the combination treatment with 1, 2, and CUR (D, F). Cell growth after a single CPT treatment and after the combination treatment with 1, 2, and CUR (E, G). Where not indicated, the Y-axis represents the percentage of cell growth compared to untreated cells. Statistical significance between single and combined treatments is presented as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
to 10 µM DOX for 2 h. In order to achieve microscopically detectable levels of fluorescence, DOX was applied at concentrations significantly higher than the IC50 values. Diarylheptanoid 1 caused partial accumulation of DOX in the cytoplasm of NCI" Fig. 4 A). The extranuclear localization H460 and HaCaT cells (l of DOX observed in NCI-H460 cells was less pronounced " Fig. 4 A). (l To test the effects of diarylheptanoids on mitochondrial structure, NCI-H460 and HaCaT cells were treated with 5 µM of diarylheptanoids for 72 h. Then, a high concentration of DOX (20 µM) was added to rapidly disrupt the mitochondria. After 2 h of DOX
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Fig. 3 Cell death induction by diarylheptanoids in cancer and normal cells. Analysis performed on untreated and NCI-H460 cells treated with increasing concentrations of 1, 2, and CUR for 72 h (A), 20 nM DOX and combinations of 10 µM of 1, 2, and CUR with 20 nM of DOX (B), untreated and HaCaT cells treated with 5 µM of 1 and 2 and 40 nM of DOX for 72 h, and combinations of 5 µM of 1 and 2 with 40 nM of DOX (C). The samples were analyzed for green fluorescence (AV, FL1-H) and red fluorescence (PI, FL2-H) by flow cytometry. (Color figure available online only.)
treatment, cells were labeled with Rho123 and imaged live " Fig. 4 B). All diarylheptanoids exhibited a protective effect on (l the mitochondrial structure. However, 1 had the most prominent effect in both cell lines. This compound preserved the tubular structure of mitochondria similar to the untreated control de" Fig. 4 B). spite subsequent DOX application (l To test superoxide anion production, NCI-H460 and HaCaT cells were treated with 5 µM of diarylheptanoids for 72 h. To assess the effect of the combinations, the cells were pretreated for 6 h with diarylheptanoids, and subsequently incubated with 2 µM CPT for an additional 72 h. Superoxide anion generation was detected by DHE staining and assessed by flow cytometric analysis " Fig. 5). The intensity of DHE in and fluorescence microscopy (l the combined treatments significantly declined in both cell lines in comparison with CPT alone. The decrease in superoxide anion production was more pronounced in HaCaT cells, with diarylhep-
" Fig. 5 A). These effects tanoid 2 exhibiting the strongest effect (l " Fig. 5 B). were confirmed by DHE imaging (l To study the mechanism behind the protective effect of diarylheptanoids observed in cells exposed to the damaging agents DOX and CPT, we analyzed the influence of diarylheptanoids on mRNA expression of enzymes with significant roles in oxidative stress, MnSOD and HIF-1α. The expression of both enzymes was analyzed in the RNA samples isolated from NCI-H460 cells, un" Fig. 6). All diaryltreated and treated with 10 µM 1, 2, and CUR (l heptanoids significantly increased the level of MnSOD mRNA ex" Fig. 6 A). Similarly, pression compared to the untreated control (l compounds 1 and 2 caused a significant increase in HIF-1α mRNA expression, while the increase observed with CUR was not statis" Fig. 6 B). tically significant (l
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Fig. 4 Diarylheptanoid 1 causes doxorubicin retention in the cytoplasm and reduces mitochondrial fragmentation induced by doxorubicin in NCI-H460 and HaCaT cells. DOX retention in the cytoplasm after diarylheptanoid 1 application in NCI-H460 and HaCaT cells (A). Subcellular localization of DOX was visualized after a 1-h incubation with 10 µM of DOX at 37 °C. Arrows point to extranuclear DOX. Nuclei were counterstained with Hoechst 33 342. Scale bar = 100 µm. Diarylheptanoid 1 reduces mitochondrial fragmentation induced by DOX in NCIH460 and HaCaT cells (B). Untreated cells possess a characteristic tubular mitochondrial structure. Mitochondrial fragmentation was induced by 20 µM of DOX for 2 h. Cells pretreated with 5 µM of 1 for 72 h were protected from mitochondrial fragmentation. Mitochondria were labeled with Rho123. Arrows point to the mitochondria which retained the tubular structure after DOX treatment. Nuclei were counterstained with Hoechst 33 342. Scale bar = 100 µm. (Color figure available online only.)
Fig. 5 Diarylheptanoid 2 decreases superoxide anion levels generated by cisplatin in NCI-H460 and HaCaT cells. Flow cytometric profiles of untreated and treated NCI-H460 and HaCaT cells evaluated after labeling with DHE (A). NCI-H460 and HaCaT cells labeled with DHE were visualized on a fluorescent microscope (B). Scale bar = 200 µm. (Color figure available online only.)
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Fig. 6 Diarylheptanoids 1 and 2 increase the level of MnSOD and HIF-1α mRNA expression in NCI-H460 cells. The effect of 10 µM of 1, 2, and CUR on MnSOD mRNA expression in NCI-H460 cells (A). The PCR products were separated on agarose gel next to a 100 bp DNA ladder. The expression of the MnSOD gene was calculated relative to β-actin expression. Statistical significance between treated and untreated (control) cells is presented as p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*). The effect of 10 µM of 1, 2, and CUR on HIF-1α mRNA expression in NCI-H460 cells (B). The PCR products were separated on agarose gel next to a 100 bp DNA ladder. The expression of the HIF-1α gene was calculated relative to gapdh expression. Statistical significance between treated and untreated (control) cells is presented as p < 0.05 (*).
Discussion !
Researchers who explore bioactive substances from plants have made increasing efforts to find safer and more efficient anticancer drugs. Particularly, plants known from folk medicine are a valuable source of new anticancer agents [21]. However, some natural sources, not considered previously, were shown to be plentiful in new bioactive substances [22]. Recently, we have explored the anticancer activity of 24 diarylheptanoids from the bark of A. glutinosa. Ten of them have not been reported previously [17]. Herein, we investigated the effects of a known platyphylloside (1) and its newly discovered analog with a p-coumaroyl group (2). Both compounds considerably inhibited the growth of chemosensitive and chemoresistant NSCLC in vitro with an IC50 < 30 µM, showing the best effect among the tested compounds [17]. Importantly, we found that diarylheptanoid 2 was less efficient in HaCaT cells, while the efficacy of both 1 and 2 was reduced in PBMCs. This implies that when applied alone, diarylheptanoids are more active in cancer cells than in normal cells. The selectivity towards cancer cells is a prerequest in choosing good drug candidates for cancer treatment. In the diarylheptanoid classic chemotherapeutics interaction study, our results demonstrated a suppressive effect of 1 and 2 on either DOX or CPT activity. Although present in cancer cells, the observed antagonism was more pronounced in normal cells. Because of this antagonism, IC50 values for DOX and CPT were sig-
nificantly raised in combination with 1 and 2. A similar effect was achieved with CUR, but it was less apparent in comparison with 1 and 2. Although our previous results suggested that diarylheptanoids could be valuable for cancer treatment, it has to be noticed that in combination with either DOX or CPT, diarylheptanoids could diminish the benefit of these drugs. On the other hand, diraylheptanoids may protect normal cells from damage caused by classic chemotherapeutics. Further, we discuss whether diarylheptanoids could be advantageous for treatment in cancer patients. The induction of apoptosis has been implicated as an important mechanism underlying cytotoxic activities of many chemotherapeutic agents. In our present study, we investigated cell death induction by diarylheptanoids in the NSCLC cell line NCI-H460. We confirmed by annexin-V/PI staining that 1, 2, and CUR significantly increase the percent of apoptotic cancer cells in a concentration-dependent manner. These results provided clear evidence of the apoptotic potential of diarylheptanoids in cancer cells. However, 1 and 2 failed to improve the DOX effect, while CUR considerably enhanced cell death induction in combination with DOX. Both diarylheptanoids diminished the effect of DOX in normal cells. Particularly, 2 decreased the cell death induced with DOX by reverting the effect to the level observed in untreated control. Considering that diarylheptanoids did not affect DOX-induced cell death in cancer cells but only failed to improve it, these compounds could be beneficial for the protection of normal cells in cancer patients. DOX accumulates in the cell nucleus intercalating into DNA inhibiting macromolecular biosynthesis [23]. The main effect is on the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription [20]. DOX also induces histone eviction from chromatin affecting the regulation of the DNA damage response in DOX-exposed cells [24]. We have observed the retention of DOX in the cytoplasm of both NCI-H460 and HaCaT cells pretreated with diarylheptanoid 1. Considering that DOX needs to enter the nucleus to reach its target molecules DNA and topoisomerase II, this finding may present one of the mechanisms behind the antagonistic interaction between 1 and DOX. However, this could be only one of the reasons for the observed antagonism of diarylheptanoids with DOX. Some authors showed that CUR might decrease topoisomerase II expression or act as topisomerase II poison [25–26], while CUR-induced topoisomerase-DNA complexes might be mediated by ROS [27]. If the diarylheptanoids that we investigated resemble the pattern of the CUR mechanism, they may antagonize the DOX effect by reducing its target availability. Although it is generally accepted that the toxicity of anticancer drugs is the consequence of their capacity to cause genomic DNA damage, they also exert important effects in other cellular compartments such as mitochondria [28]. One of the mechanisms proposed to be responsible for the toxicity of DOX is the generation of ROS, which mostly affects the mitochondria function [29]. One µM of DOX was enough to disrupt the tubular structure of mitochondria in H9c2 myoblast cells after 24 h treatment [30]. We have shown that DOX treatment increased mitochondrial fragmentation in NCI-H460 and HaCaT cells, which was illustrated by Rho 123 staining. When cells were pretreated with 1, the DOX effect was attenuated, leading to the mitochondrial structure recovering. Particularly under these conditions, the mitochondria structure resembles the structure observed in untreated cells. Other authors showed that antioxidants suppress
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Original Papers
the mitochondrial transmembrane potential decrease associated with the DNA damaging agentʼs action [31]. Therefore, we assumed that diarylheptanoids could scavenge ROS and act as antioxidants antagonizing DOX and CPT effects. It is known that ROS plays an important role in CPT cytotoxicity [19]. We compared the effect of 2 µM CPT alone and in combination with 10 µM of 2 on superoxide anion production in NCIH460 and HaCaT cells. CPT in this concentration exerted significant inhibition of cell growth in both the cancer and non-transformed cell lines. Consequently, we observed the increase at the superoxide anion level after CPT application. In cells pretreated with 2, superoxide anion production was efficiently decreased, suggesting that the diarylheptanoids capacity to antagonize CPT cytotoxicity is due to its ROS scavenging ability. Importantly, the reversal effect of 2 on ROS levels was irrelevant in cancer cells and pronounced in normal human keratinocytes, suggesting that the chemoprotection effect is only in normal cells. Hypoxia or pretreatment with antioxidant agents may attenuate death induction by DNA damaging drugs [32]. Therefore, we have examined the effects of 1 and 2 on Mn-SOD and HIF-1α mRNA levels in cancer cells. Superoxide dismutases are enzymes which catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide, and represent an important antioxidant defense in cells exposed to high levels of oxygen [33]. Mn-SOD is present in mitochondria and peroxisomes. HIF-1, controlled by the rapid stabilization of the HIF-1α subunit, is an essential transcriptional factor in the cellular and systemic responses to hypoxia [34]. Both enzymes have an impact on the intracellular oxidative status leading to the efficient elimination of ROS. We found that 1 and 2 significantly increased the expression of Mn-SOD and HIF-1α mRNA. In comparison with CUR, the effect of 1 and 2 was more apparent. These results suggest that diarylheptanoids could be considered anticancer agents with antioxidant properties. Treatment with a DNA topoisomerase inhibitor, such as DOX, and with an alkylating agent, such as CPT, may cause ROS accumulation in cancer and non-transformed normal cells. These effects could be suppressed by the cytoprotective action of diarylheptanoids found in the present investigation. These compounds may contribute to the protection of normal cells, which are sensitive to the formation of free radicals in the course of chemotherapy. However, their use cannot improve the efficacy of anticancer drugs. Further studies are necessary, in this respect, to confirm diarylheptanoids as candidates for treatment in cancer patients.
Chemicals RPMI 1640 medium, DMEM medium, penicillin–streptomycin solution, antibiotic–antimycotic solution, L-glutamine, and trypsin/EDTA were purchased from PAA. FBS, Rho123, and DMSO were obtained from Sigma-Aldrich Chemie GmbH. PI and AV were purchased from Abcam. DHE was obtained from Molecular Probes®, Invitrogen.
Cell culture The NCI-H460 cell line was purchased from the American Type Culture Collection. NCI-H460 cells were maintained in RPMI 1640 supplemented with 10 % FBS, 2 mM L-glutamine, 4.5 g/L glucose, 10 000 U/mL penicillin, 10 mg/mL streptomycin, and 25 mg/mL amphotericin B solution at 37 °C in a humidified 5 % CO2 atmosphere. The HaCaT cell line (normal human keratinocytes obtained from CLS – Cell Lines Service) was a generous gift from Prof. Andra Jorg, Division of Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany. HaCaT cells were cultured in DMEM supplemented with 10% FBS, 4 g/L glucose, L-glutamine (2 mM), 5000 U/ml penicilin, and 5 mg/mL streptomycin solution.
Isolation of peripheral blood mononuclear cells Heparinized blood taken from healthy volunteers and PBMCs were isolated by centrifugation in lymphocyte separation medium (Ficoll, ICN Biomedicals), according to the manufacturerʼs instructions.
Cytotoxicity by the MTT assay Cell viability was assessed by the MTT assay (Sigma). Briefly, cells were seeded in 96-well microplates and treated with 1, 2, DOX, and CPT for 72 h. In combined treatments, cells were pretreated with 5, 10, and 25 µM of 1, 2, or CUR at 37 °C for 6 h and subsequently treated with increasing concentrations of DOX or CPT. The absorbance of formazan dye was measured at 540 nm using an automatic microplate reader (LKB 5060–006 Micro Plate Reader). Growth inhibition (I) was determined according to the following equation: I (%) = [1 − (Atreated sample/Auntreated control)] × 100 where A is absorbance. The IC50 value was defined as the concentration of each drug that inhibited cell growth by 50%. The IC50 was calculated by linear regression analysis using Excel software.
Materials and Methods !
Median effect analysis
Drugs
The nature of the interaction between different drugs was analyzed using CalcuSyn software (Biosoft) that uses the CI method [35], based on the multiple drug effect equation. At least three data points were used for each single drug in each designed experiment. The non-constant ratio combination was chosen to assess the effect of both drugs in combination. The results are presented by a fraction-affected CI graph. Values of CI < 1 point to a pronounced additive effect or synergism, i.e., the smaller the value, the greater the degree of synergy. A value of CI = 1 indicates an additive effect, and values of CI > 1 point to an antagonistic effect.
DOX solution was obtained from Ebewe Arzneimittel GmbH. CPT was obtained from Pfizer (Perth) Pty Ltd. Both drugs were diluted in sterile water as was prescribed for human use. CUR was purchased from Sigma-Aldrich Chemie GmbH and diluted in absolute ethanol. According to the manufacturer, the purity of CUR was ≥ 98.0 % (HPLC). Diarylheptanoids 1 and 2 were isolated from A. glutinosa bark, as described previously [17]. The purity of tested compounds 1 and 2 was higher than 98 % as determined by HPLC‑DAD (280 nm) and 1H NMR [17, 18].
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Cell death detection The percentages of apoptotic, necrotic, and viable cells were determined by AV and PI labeling. NCI-H460 cells were incubated overnight in 6-well plates and subjected to single treatments with 10, 25, and 50 µM of 1, 2, and CUR. In combined treatments, NCI-H460 cells were pretreated with 10 µM of 1, 2, or CUR, while HaCaT cells were pretreated with 5 µM of 1 or 2 at 37 °C for 6 h and subsequently treated with 20 nM or 40 nM DOX, respectively. After 72 h, the attached and floating cells were collected by centrifugation. AV/PI staining was performed according to the manufacturerʼs instructions and cells were analyzed within 1 h by flow cytometry. The fluorescence intensity (green FL1-H and red FL2-H) was measured on a FACSClibur flow cytometer (Becton Dickinson). In each sample, 10 000 cells were recorded (gated to exclude cell debris), and the percentages of viable (AV- PI-), early apoptotic (AV+ PI−), apoptotic and necrotic (AV+ PI+), and already dead (AV− PI+) cells were analyzed by CellQuest Pro data analysis software.
Superoxide anion detection Flow cytometric analysis of DHE fluorescence intensity was used to detect the superoxide anion level in cells. NCI-H460 and HaCaT cells were incubated overnight in 6-well plates, then pretreated with 5 µM of 1, 2, and CUR at 37 °C for 6 h and subsequently treated with 2 µM CPT. After 72 h, adherent cells were harvested by trypsinization and incubated in adequate medium with 1 µM DHE for 30 min at 37 °C in the dark. Cells were subsequently washed twice in PBS, and DHE fluorescence was analyzed by flow cytometry (excitation 488 nm and emission 585 nm, FL2-H channel). MFI was calculated after correction for autofluorescence.
on a spectrophotometer, and quality was determined by agarose gel electrophoresis. RT reactions using 2 µg of total RNA were performed with a high-capacity cDNA reverse transcription kit (Applied Biosystems), following the manufacturerʼs instructions. PCR reactions were performed with primers specific for MnSOD and HIF-1α [36–37]. β-actin [38] and gapdh [39] were used as internal controls and coamplified with MnSOD and HIF-1α, respectively. The PCR reactions for MnSOD were performed on a GeneAmp® PCR System 9700 (Applied Biosystems) under the following conditions: initial denaturizing at 94 °C for 5 min, 27 cycles at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, and at 4 °C indefinitely. For the amplification of HIF-1α, 25 cycles were performed and the annealing temperature was decreased to 56 °C. The gapdh primers were used at a 1 : 5 ratio to HIF-1α primers in order to attain linear amplification conditions. The β-actin primers were used at a 1 : 1 ratio to MnSOD primers in order to attain linear amplification conditions. The PCR products were separated in 2 % agarose gels stained with ethidium bromide. MultiAnalyst/PC Software Image Analysis (Bio-Rad Gel Doc 1000) was employed for densitometry analysis.
Statistical analysis Statistical analysis was performed by Statistica 6.0 software. The differences between groups were examined by the Studentʼs ttest. Statistical significance was accepted if p < 0.05.
Supporting information The IC50 values of compounds 1, 2, and CUR as well as classic chemotherapeutics DOX and CPT, in single and combined treatments with diarylheptanoids, in NCI-H460 and HaCaT cells and PBMCs are available as Supporting Information.
Fluorescence microscopy NCI-H460 and HaCaT cells were seeded in 8-chamber slides (Nunc) or glass bottom petri dishes and allowed to grow at 37 °C overnight before treatment. To detect superoxide anion, cells were washed in PBS and labeled with 1 µM DHE, a superoxide indicator dye, for 30 min at 37 °C. Live NCI-H460 cells were examined under a Zeiss Axiovert fluorescent microscope (Carl Zeiss Foundation) using AxioVision 4.8 software. HaCaT cells were analyzed under a Leica fluorescent microscope (Leica Microsystems DMIL) using Leica Application Suite software (version 2.8.1). To evaluate changes in the mitochondrial structure, cells were washed in PBS and labeled for 30 min at 37 °C with 5 µM of Rho123, which accumulates in the mitochondria of living cells. Nuclei were counterstained with Hoechst 33 342 (Sigma-Aldrich Chemie) for 15 min at room temperature, and live cells were examined under a Zeiss Axiovert fluorescent microscope. To examine subcellular localization of DOX, cells were incubated with 10 µM of DOX for 1 h at 37 °C. After washing in PBS, nuclei were counterstained with Hoechst 33 342 for 15 min at room temperature and cells were fixed with 4 % paraformaldehyde. Subcellular localization of DOX, which possesses inherent red fluorescence, was analyzed under a Zeiss Axiovert fluorescent microscope.
RNA extraction and reverse transcription polymerase chain reaction Total RNA was extracted from untreated NCI-H460 cells and cells treated with 10 µM of 1, 2, and CUR at 37 °C for 72 h. The isolation was carried out using Trizol (Invitrogen Life Technologies), according to the manufacturerʼs instructions. RNA was quantified
Acknowledgements !
This research was supported by the Ministry of Education, Science and Technological Development of Serbia (grant nos. III 41031 and III 41025).
Conflict of Interest !
The authors declare no conflict of interest
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