Abstract
Background/Aim: Osthole is a simple coumarin that has been found to have anticancer, anti-inflammatory, antiviral, anticoagulant, anticonvulsant and antiallergic activities. The aim of this study was to analyze the combined anti-proliferative effect of cisplatin (CDDP) and osthole on a rhabdomyosarcoma cell line, and assess the pharmacology of drug-drug interaction between these drugs using isobolographic analysis. Materials and Methods: The anticancer actions of osthole in combination with CDDP were evaluated using the tetrazolium dye-based MTT cell proliferation assay. Results: Osthole and CDDP applied together augmented their anti-cancer activities and yielded an additive type of pharmacologic interaction by means of isobolographic analysis. Conclusion: Combined therapy using osthole and cisplatin could be suggested as a potential chemotherapy regimen against rhabdomyosarcoma.
Cis-diamminedichloroplatinum (II) (Cisplatin, CDDP) is a widely used chemotherapeutic agent in the treatment of various neoplasias. CDDP is considered the mainline treatment of cancers of the ovaries, testes, and solid tumors of the head and neck (1). Despite its toxic side-effects, including severe kidney problems, gastrointestinal disorders, hemorrhage, and hearing loss, CDDP is also used against cancers of soft tissue, bones (2), muscles, and sarcomas (3), including rhabdomyosarcoma (RMS) (4). RMS originating from mesenchymal stem cells develop malignant tumors of soft tissues located often in the head/neck and genitourinary system in children and adolescents causing aggressive disease and clinical complications (5). Patients diagnosed with RMS have a poor prognosis, which is caused by late diagnosis, metastasis and local recurrence (6). Most patients with RMS are treated by surgery, radiotherapy and chemotherapy consisting of vincristine, actinomycin and cyclophosphamide (VAC) as standard therapy. Although many other chemotherapy regimens (vincristine, topotecan and cyclophosphamide -VTC, etoposide, ifosfamide and vincristine - IEV, actinomycin. etoposide and vincristine - DEV) have been applied, no significant statistical relationship between protocol of treatment and RMS outcome has yet been reported (7). Slightly better results were observed for protocols consisting of vincristine, cyclophosphamide and cisplatin (VCP) (4), suggesting that CDDP may augment RMS chemotherapy. Nevertheless, RMS treatment outcome has not been improved for three decades (5). Thereby, better novel approaches or additive treatments are needed.
In our previous study, we demonstrated that CDDP inhibits RMS cells (TE671 cell line) proliferation, especially when combined with histone deacetylase inhibitors (8). On the other hand, we also reported that simple coumarin osthole (7-methoxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one), a natural product derived from medicinal plants display anti-cancer activity against RMS TE671 cell line by inhibiting their proliferation, inducing apoptosis, and slowing down the cell cycle progression, presumably trough increase of TP53 and CDKN1A genes expression (9). Osthole has also been reported to inhibit migration and invasiveness of several other cancer cell lines, including breast cancer cells (10), lung adenocarcinoma (11), hepatocellular carcinoma (12), glioma (13), and osteosarcoma (14). In the present study, we combined osthole and CDDP in an experimental treatment against RMS cells in order to assess their pharmacological interaction by means of advanced isobolographic analysis.
Anti-proliferative effects of CDDP and osthole (OST) administered singly in the TE671 cancer cell line.
Materials and Methods
Cell culture. TE671, human rhabdomyosarcoma cell line was obtained from the European Collection of Cell Cultures (ECACC). Mycoplasma free cells were routinely maintained in Dulbecco's modified Eagle's medium F12 Ham (Sigma – Aldrich, St. Louis, MO, USA) supplemented with 10% v/v fetal bovine serum and antibiotics penicillin (100g/ml) and streptomycin (100g/ml) (Sigma – Aldrich, St. Louis, MO, USA) incubated at 37°C in humidified atmosphere containing 5% CO2.
Cell treatments. Cisplatin (Sigma – Aldrich, St. Louis, MO, USA), and osthole were dissolved in phosphate buffered saline (PBS) with Ca2+ and Mg2+, or in methanol as stock solutions, respectively. Osthole (OST) was isolated from fruits of Mutellina purpurea using high-performance counter-current chromatography (HPCCC) as we described previously in detail (9). TE671 cells (1×104 cells/well) were seeded into 96-well cell culture plates for 24h at 37°C. Next day, cells were incubated with increasing concentrations of osthole (0, 405-4,86 μg/ml) and cisplatin (0,06-0,71 μg/ml) for 96 h.
MTT assay. Inhibition of cancer cells proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay as we described previously (8). After treatment with the examined compounds the cells were incubated with 10 μl of MTT solution (5 mg/ml, Sigma – Aldrich, St. Louis, MO, USA). The reaction was stopped after 3 using 10% SDS in 0,01N HCl solution. Finally, absorbance was measured at 570 nm (Infinite M200 Pro Microplate Reader, Tecan, Männedorf, Switzerland).
Type I isobolographic analysis of interaction between CDDP and OST at the fixed-ratio combination of 1:1 in the cancer cell line TE671 measured in vitro by the MTT assay.
Isobolographic analysis. Isobolographic analysis- a statistical method allowing the precise characterization of pharmacodynamic interaction between drugs (15) was performed as described previously (8, 16-18), First, the percent inhibition of cell viability per increasing doses of CDDP, and osthole administrated singly in the rhabdomyosarcoma cell line TE671 was measured. Subsequently, the dose-response effects for each investigated anti-cancer compound (i.e., CDDP, osthole) were fitted with log-probit linear regression analysis as described by Litchfield and Wilcoxon (1949) (19). The test for parallelism of dose-response effects for CDDP and osthole, revealed that CDDP had its dose-response effect non-parallel to that of osthole in the rhabdomyosarcoma cell line (TE671) measured by the MTT assay. The type of interactions between CDDP and osthole in the cancer cell line TE671 was isobolographically analyzed according to the methodology described elsewhere (16, 20). From the experimentally denoted IC50 values for the drugs administered alone, median additive inhibitory concentrations of the mixture of CDDP with osthole at the fixed-ratio of 1:1 (IC50 add) – i.e., concentrations of the mixture, which should theoretically inhibit cell viability in 50% were calculated as described earlier (16). Subsequently, the experimentally-derived IC50 mix at the fixed-ratio of 1:1 was determined based on the concentration of the mixtures of CDDP with osthole, inhibiting 50% of cell viability in the cancer cell line (TE671) measured in vitro by the MTT assay. The separate concentrations of CDDP and osthole in the mixture were calculated from, the IC50 mix values by multiplying this value by the respective proportions of particular drugs. Additional information concerning the isobolographic analysis has been published elsewhere (16, 20).
TE671 cells were incubated with 1:1 drug mixture (CDDP/osthole) in increasing concentrations for 96 h showing a dose-dependent decrease of cancer cell viability (MTT test). Each bar represents mean optical densities±S.E.M., (***p<0.001 versus control; Student's test) of 24 measurements from three independent experiments.
Statistical analysis. GraphPad Prism software was used for data analyzing (One-way ANOVA with Tukey post hoc test). p<0.05 was considered to indicate a statistically significant difference. Results were presented as mean±standard error of the mean (SEM).
Log-probit analysis was used to determine the experimentally-derived IC50 and IC50 mix values for CDDP, and osthole, when the drugs were administered alone or in combination for the fixed-ratio of 1:1 (19). Difference between the experimentally-derived IC50 mix values for the mixture of CDDP with osthole and the theoretically additive IC50 add values was statistically verified by using the unpaired Student's t-test, as presented elsewhere (20).
Results
As we demonstrated previously, CDDP (8) and osthole (9) inhibited TE671 cells proliferation in a dose-dependent manner when applied separately. In the present study using Log-probit dose-response analysis we determined IC50 for osthole (Table I), whereas IC50 for CDDP is 0.591±0.187, as we demonstrated previously (8). Next, we evaluated the anti-proliferative effects of osthole administered in combination with CDDP to the TE671 cell line. Incubation of RMS cells with different concentrations of both drugs based on established IC50 values resulted in dose-dependent reduction of cancer cells viability (Figure 1).
The test for parallelism of dose-response effects between CDDP and osthole confirmed that the log-probit lines of these compounds were non-parallel to one another (Table I; Figure 2).
The combinations of CDDP with osthole (at the fixed-ratio of 1:1) produced the definite anti-proliferative effects in the TE671 cell line and the experimentally-determined IC50 mix values for the two-drug mixture were 3.568±0.555 μg/ml for the combination of CDDP with osthole (Table II, Figure 3). With type I isobolographic analysis, no statistical difference was observed between the IC50 mix and IC50 add values with unpaired Student's t-test and thus, the analyzed interactions between CDDP and osthole were additive (Table II).
Discussion
Chemotherapy of rhabdomyosarcoma remains an oncological challenge. VAC (vincristin, adriamycin plus cyclophosphamide) has been considered the gold standard regimen for RMS. A treating regime using many individually active agents (e.g. doxorubicin, cisplatin, etoposide, ifosfamide, topotecan, melphalan) failed to improve significantly outcomes as compared to VAC (4). Thereby, novel approaches or combinations of drugs that result in an additive treatment are needed.
Log-probit dose-response relationship curves (DRRCs) for cisplatin (CDDP) and osthole (OST) administered alone, and in combination at the fixed-ratio of 1:1 (in red), illustrating the anti-proliferative effects of the drugs in the cancer cell line (TE671) measured in vitro by the MTT assay. Doses of CDDP and OST administered separately and the mixture of the drugs at the fixed-ratio combination of 1:1 (in red) were transformed into logarithms, whereas the anti-proliferative effects produced by the drugs in the cancer cell line (TE671) measured in vitro by the MTT assay were transformed into probits according to Litchfield and Wilcoxon (1949) (19). Linear regression equations of DRRCs are presented on the graph; where y: is the probit of response, and x: is the logarithm (to the base 10) of a drug dose, R2: coefficient of determination. Test for parallelism revealed that the experimentally determined DRRCs for CDDP and OST (administered alone) are not parallel to one another (for more details see Table I).
Isobologram showing additive interaction between cisplatin (CDDP) and osthole (OST) with respect to their anti-proliferative effects in the cancer cell line TE671, measured in vitro by the MTT assay. The median inhibitory concentrations (IC50) for CDDP and OST are plotted on the X- and Y-axes, respectively. The solid lines on the X and Y axes represent the S.E.M. for the IC50 values for the studied drugs administered alone. The lower and upper isoboles of additivity represent the curves connecting the IC50 values for CDDP and OST administered alone. The dotted line starting from the point (0, 0) corresponds to the fixed-ratio of 1:1 for the combination of CDDP with OST. The diagonal dashed line connects the IC50 for CDDP and OST on the X- and Y-axes. The points A’ and A” depict the theoretically calculated IC50 add values for both, lower and upper isoboles of additivity. The point M represents the experimentally-derived IC50 mix value for total dose of the mixture expressed as proportions of CDDP and OST that produced a 50% anti-proliferative effect in TE671 as measured in vitro by the MTT assay. On the graph, the S.E.M. values are presented as horizontal and vertical error bars for every IC50 value. Although the experimentally-derived IC50 mix value is placed above the point A”, statistical analysis of data revealed no significant differences among IC50 mix and IC50 add values. Thus, additive interaction exists between CDDP and OST in the cancer cell line TE671, as measured in vitro by the MTT assay.
Osthole has reportedly shown anticancer activity against several cancer cell types both in vitro (10, 11, 21) and in vivo (12) including rhabdomyosarcoma cells (9). Osthole's mechanism of action has been attributed to the inhibition of cell cycle progression, induction of apoptosis (9) interfering with cancer survival signaling pathways, including AKT and ERK kinases, as well as attenuating matrix metalloproteinases activity (21). On the other hand, osthole displays several protective properties, including osteogenic and immuno-modulatory activities, as well as hepatoprotective and neuroprotective actions (21). In the context of anticancer therapy, causing often severe toxic side effects, the simultaneous use of osthole and CDDP seems to have a beneficial influence potentially ameliorating some conventional drug-induced side-effects. However, this issue requires further in vivo studies.
CDDP, an old drug, has recently received renewed attention, especially in the treatment of heterogeneous, rapidly proliferating cancer types, including triple-negative (lacking estrogen receptor, progesterone receptor, and HER expression) breast cancer cells (TNBC) (22). RMS cells are characterized by very fast, uncontrolled proliferation, and poor outcome for RMS-bearing patients (5). Thereby, in the present work we performed an experimental treatment of RMS cells combining osthole and CDDP in vitro. Using isobolographic analysis for the precise assessment of pharmacological drug-drug interaction type utilizing several combinations of both drugs we were able to show additive interaction of osthole and CDDP, resulting in a greatly enhanced anticancer effect of these drug combination compared to single drug treatment. Similar results we presented also recently combining CDDP and histone deacetylase inhibitors against RMS cells (8). This kind of combination therapy could be very promising in overcoming cisplatin resistance. Additionally, application of osthole decreased the doses of cisplatin used to achieve the same treatment efficiency and potentially ameliorating some of the CDDP-induced side effects. Combination therapy of osthole (23) or its derivative (24) and CDDP was also reported in other single reports, however usually very limited number of drug concentrations were examined, sometimes only one (23), which does not allow to establish a drug-drug type of pharmacokinetic interaction. In our present detailed study, using several CDDP/osthole combinations we were able to clearly demonstrate an additive interaction in RMS cells.
Beneficial effects of CDDP/osthole treatment have also been observed against NCI-H460 lung cancer cells in vitro (23), showing that this combination could be regarded as a general phenomenon and rather not cancer cell type-specific. This could support the potential role of osthole as a drug candidate in cancer treatment.
- Received October 19, 2017.
- Revision received October 31, 2017.
- Accepted November 1, 2017.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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