The Combination of Amygdalin with Some Anticancer,Antiparasitic,and Antigout Drugs Against MG63,Saos2,SW1353,and FL Cells In Vitro

Abstract

Osteosarcoma has a poor prognosis and survival rate due to inadequate chemotherapy, high recurrence ability, high metastasis potential, and almost no radiotherapy being applied. One of the strategies to solve these problems is to develop the pharmacologically active plant metabolite, amygdalin, in combination therapeutic systems. In this project, the antiproliferative effects of amygdalin alone and in binary or ternary combinations with some anticancer drugs (cisplatin, 5-fluorouracil, oxaliplatin, and camptothecin), antiparasitic drugs (metronidazole and miltefosine), and an antigout drug (colchicine) were examined using human bone osteosarcoma cell lines (MG-63 and Saos2), the chondrosarcoma cell line (SW1353), and the normal human cell line (FL). Known half-maximal inhibitory concentration values of the drugs were taken into consideration, and the recommended combination ratios were used in the Chou–Talalay method. The strong synergistic effect commonly seen in the combination of amygdalin with miltefosine, metronidazole, camptothecin, colchicine, oxaliplatin, 5-fluorouracil, and cisplatin dual drug indicates that these combinations can be used in cancer treatment. The synergistic effect caused by amygdalin decreases toxicity by increasing drug yield. However, amygdalin antagonism seen in several combinations may prevent these pairs from being used together. In combination with antagonistic effects, it may be preferable to use amygdalin alone as it generally causes strong antiproliferative effects. Besides, there is a more potent synergism between amygdalin and triple drug combinations. Overall, these results emphasize that amygdalin combinations in treatment of bone cancer are significant.

Introduction

Despite advances in radiotherapy and chemotherapy, osteosarcoma has a poor prognosis and survival rate because almost no radiotherapy is applied, response to chemotherapy is poor, and it has a high recurrence ability and high metastasis potential. One of the strategies to solve these problems is to develop new therapeutic systems. Combined treatment modalities with effective drug molecules have resulted in high clinical outcomes. Cyanogenic glycosides, critical plant secondary metabolites, are among the candidate molecules that can be used in new anticancer drug development studies due to their very different structural properties, which may enter into unique interactions with biomolecules in their spatial arrangements. Amygdalin, a cyanogenic glycoside, has been used in pharmacological studies for many years. Following ingestion of amygdalin by mouth, it is degraded into highly toxic cyanide along with other by-products by digestive enzymes.¹ This cyanide is detoxified by rhodanese, which is abundant in normal cells. However, amygdalin has long been tested for its use in treatment of many diseases, including chronic alcoholism and hemorrhoids.² Amygdalin can kill tumor cells by causing hydrogen cyanide (HCN) formation and it has been reported that this molecule could therefore be used as an anticancer drug.³ HCN shows its toxic effect by stopping adenosine triphosphate production and oxygen use as a result of binding of the electron transport chain in mitochondria to the last enzyme.⁴ This can be used to selectively destroy cancer cells since the enzyme, rhodanese, which provides detoxification of cytotoxic HCN and is released into the environment as a result of breakdown of amygdalin, is less common in cancer cells. However, there are reports in the literature that question the usefulness of amygdalin in the treatment of cancer. Soffer reported that the use of laetrile in treatment of neoplasms is useless and, at the same time, dangerous because it can delay the correct treatment options and is toxic.⁵ Hill et al. reported that applying parenteral amygdalin on C57BL/6 mice bearing B16 melanoma and BW5147 AKR leukemia resulted in ineffective treatment.⁶ Chitnis et al. noticed that administration of amygdalin did not increase the survival time of BDF1 mice bearing P388 lymphocytic and P815 mast cell leukemia.⁷ However, Fukuda et al. stated that the amygdala has a protective effect on cells by inhibiting tumor formation.⁸ There are also reports that amygdalin can provide pain relief and prevent cough.^9
–11 Although the Food and Drug Administration (FDA) concluded that the amygdalin molecule was ineffective and toxic as a result of studies carried out from the end of the 1970s to the present day, it has still attracted the attention of many modern scientists.¹² Recently, significant progress has been made in the development and transfer of amygdaline and semisynthetic formulations.¹³

Materials and Methods

In this project, all 5-FU chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The cell lines were kindly provided by Prof. Hasan Havitcioglu (Dokuz Eylul University). The antiproliferative effects of amygdalin alone and in binary or ternary combinations with some anticancer drugs (cisplatin, 5-fluorouracil, oxaliplatin, camptothecin), antiparasitic drugs (metronidazole and miltefosine), and an antigout drug (colchicine) were examined using two human bone osteosarcoma cell lines (MG-63 [ATCC^® CRL-1427™] and Saos-2 [ATCC HTB-85™]), one human bone chondrosarcoma cell line (SW-1353 [ATCC HTB-94™]), and one normal cell line (FL [ATCC CCL-62™]). The data obtained from the MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] test were analyzed with CalcuSyn software, version 2.11, commonly used to study drug interactions described by Chou¹⁴ and Chou and Talalay.¹⁵ The antiproliferative effects of amygdalin with these conventional chemotherapeutic agents were studied for the first time on the MG63, Saos2, SW1353, and FL cell lines.

The MG-63 and FL cell lines were maintained in Eagle's minimum essential medium (Corning) supplemented with 10% fetal bovine serum. The SW-1353 cell line was maintained in Dulbecco's modified Eagle's medium/F12 (Corning) supplemented with 10% fetal bovine serum. McCoy's 5A medium (Corning) modified and supplemented with 15% fetal bovine serum was used for the Saos-2 cell line. As an antibiotic, Pen-Strep solution (10,000 U/10 mg) (Sigma-Aldrich) was preferred for all of the cells. At confluence in the log phase, cells were removed from the flasks using 4 mL of 0.25% trypsin-EDTA solution (Sigma-Aldrich) using a centrifugation process, and then the cell pellet was dissolved in 3 mL of fresh modified medium. A cell suspension containing ∼1 × 10⁴ cells in 100 μL was seeded into the wells of 96-well culture plates. The cells were treated with the drugs dissolved in sterile dimethyl sulfoxide (DMSO) (max 0.5% of DMSO) (see Supplementary Table S1 for final concentrations) at 37°C with 5% carbon dioxide (CO₂) for 24 h. The final volume of the wells was set to 200 μL by the medium. To obtain the optimal combination ratio, the half-maximal inhibitory concentration (IC₅₀) of these drugs was used and the recommended equipotency ratios are shown in Supplementary Table S2 (for detailed information, CalcuSyn reports can be found in Supplementary Data). Known IC₅₀ values of the drugs for obtaining the optimum combination ratio for maximal synergy were taken into consideration and the recommended combination ratios were used in the Chou–Talalay method. According to this information, to form an equipotency ratio, the doses 0, 1/4, 1/2, 1, 2, and 4 of IC₅₀ of each drug were used. Therefore, for a three-drug combination (A, B, and C), their IC₅₀ ratios a:b:c [e.g., (IC₅₀)A/(IC₅₀)B/(IC₅₀)C ratio] were used. For two-drug combinations that were conducted at the same time, the ratios used were, for example, A + B (a:b), B + C (b:c), and A + C (a:c). The MTT cell proliferation assay for different binary and ternary drug combinations was used to evaluate in vitro pharmacodynamic drug interaction analysis results for the selected drugs. The cell proliferation assay was evaluated by the MTT (yellow tetrazolium MTT) method.¹⁶ Absorbance data (CLARIOstar microplate reader) were loaded for automated calculation of the slope of the median-effect plot (m), the dose that produces 50% effect such as IC₅₀ (Dm), and the linear correlation coefficient of the median-effect plot (r) parameters, as well as the combination index (CI) and dose reduction index (DRI) using CalcuSyn software, version 2.11. The cytotoxicity of compounds was also determined through a lactate dehydrogenase assay. Approximately 5 × ∼10³ cells in 100 μL were placed into 96-well plates as triplicates and treated with IC₅₀ concentrations of test compounds at 37°C with 5% CO₂ for 24 h. Lactate dehydrogenase activity was obtained by determining absorbance at 492–630 nm using a microplate reader. The cytotoxicity assay results were noted as percent cytotoxicity according to the following formula: percentage of cytotoxicity = [(experimental value – low control / high control – low control) × ∼100].

Results

Single-drug cell proliferation assay

After performing the MTT cell proliferation assay for each drug alone against the cells, the CalcuSyn software was used for both generation of single-drug dose–effect curves and calculation of the mass action law parameters (Dm), (m), and (r). As shown in Supplementary Table S4a, all the drugs inhibited cell growth in a dose-dependent manner. The Dm values (similar to IC₅₀) of tested drugs were found to range from 1.09E+08 to 7.33E+18 μM in FL cells. When the Dm values of these drugs were examined in the same table, the highest to lowest order of their potency in FL cells was CIS > CPT >5FU > OX > MTZ > COL > AMY > MTF. In MG63 cells, Dm values (similar to IC₅₀) of tested drugs were found to be between 7.97E+03 and 4.46E+08 μM. The potency (Dm values) of these drugs was ordered from large to small as follows: AMY >5FU > CPT > MTZ > OX > CIS > COL > MTF. When the Dm values of these drugs were examined in the same table for Saos-2 cells, it was understood that Dm values were between 5.37E+04 and 2.17E+18 μM and their potency (Dm values) order was CPT > AMY >5FU > OX > COL > CIS > MTZ > MTF. In SW-1353 cells, Dm values (similar to IC₅₀) of tested drugs were found to be between 6.01E+02 and 4.46E+08 μM. The potency (Dm values) of these drugs was ordered from large to small as follows: AMY > CPT > MTF > CIS >5FU > MTZ > COL > OX. As shown in Supplementary Table S4a, the dose–effect curves of the tested drug on the cells were flat sigmoidal (m < 1) or sigmoidal (m > 1). The median-effect plots of all tested drugs are shown in Supplementary Table S4a. All the (r) values of the curves were above 0.95, indicating acceptable conformity with the mass action law. When the cytotoxic analysis results of the compounds were examined, they were found to have percent cytotoxicity values of 7–21% (Supplementary Table S7).

Cell proliferation assay of binary and ternary drug combinations

The results of the single-drug cytotoxicity assay met the preconditions of the Chou–Talalay method for initiating the in vitro pharmacodynamic drug interaction analysis. Therefore, we tested all possible combinations of binary and ternary drugs in a fixed-rate combination design. All curves were flat sigmoidal (m < 1) or sigmoidal (m > 1), with r above 0.95. The parameters (m, Dm, and r), CIs, and the fraction affected (fa)-DRI table for amygdalin combinations are presented in Supplementary Tables S4a, S5, and S6, respectively. The polygonograms at fa = 0.5, 0.75, and 0.9 present the experimental demonstration of the interaction between amygdalin and other drugs (Supplementary Fig. S1). The Chou–Talalay method for drug combination is based on the median-effect equation, which provides the theoretical basis for the CI that allows quantitative determination of drug interactions, where CI <1, CI = 1, and CI >1 indicate synergism, additive effect, and antagonism, respectively (Supplementary Table S3).¹⁷

First, the present study demonstrates that the amygdalin dose (AMY) and colchicine dose (COL; 7.02E−01) or metronidazole dose (MTZ; 1.48E–01) for MG63 cells showed a moderate synergistic (0.7 < CI <0.85) and a strong synergistic effect (0.1 < CI <0.3) at fa = 0.5 (Supplementary Table S5), respectively. The combination of AMY and cisplatin dose (CIS) or 5-fluorouracil dose (5FU) or oxaliplatin dose (OX) has a very strong synergistic effect, with CI values ranging from 3.81E–03 to 1.79E–03 for fa = 0.5, as noted in the fa-CI plot (when MG63 cell growth was inhibited by 50%) (Supplementary Table S5). The association of AMY and camptothecin dose (CPT) for MG63 cells resulted in a strong antagonistic effect (3.3 < CI <10). The combination of AMY and miltefosine dose (MTF) caused a very strong antagonistic effect (CI >10) at fa = 0.5 in MG63 cells (Supplementary Table S5). The combination of AMY and CIS (9.50E–01) or MTF (1.85E–01) for FL cells showed a nearly additive effect (0.90 < CI <1.10) and a strong synergistic effect (0.1 < CI <0.3) at fa = 0.5 (Supplementary Table S5), respectively. The combination of AMY and 5FU (7.08E+00) for FL cells caused a strong antagonistic effect (3.3 < CI <10) at fa = 0.5. However, the combination of AMY and other molecules for FL cells exhibited a very strong antagonistic effect (CI >10) at fa = 0.5. In Saos2 cells, the synergism between AMY and 5FU or COL or CPT is very strong, with CI <0.1 for fa = 0.5. Interestingly, the antagonism between AMY and CIS or MTZ for Saos2 cells is very strong, with CI >10 for fa = 0.5 (Supplementary Table S5). The combination of AMY and OX (1.22E+00) or MTF (6.89E+00) for Saos2 cells showed a moderate antagonistic (1.20 < CI <1.45) and a strong antagonistic effect (3.3 < CI <10) at fa = 0.5 (Supplementary Table S5), respectively. In SW1353 cells, the combination of AMY and CIS caused a synergistic effect (0.3 < CI <0.7) at fa = 0.5 (Supplementary Table S5). The synergism between AMY and CPT for SW1353 cells is very strong, with CI <0.1 for fa = 0.5 (Supplementary Table S5). The combination of AMY and COL (4.48E+00) or MTZ (2.25E+00) for SW1353 cells showed a strong antagonistic (3.3 < CI <10) and an antagonistic effect (1.45 < CI <3.3) at fa = 0.5 (Supplementary Table S5), respectively. The combination of AMY and 5FU or OX or MTF caused a very strong antagonistic effect (CI >10) at fa = 0.5 in SW1353 cells (Supplementary Table S5). It can be seen that AMY triple combinations with a fractional inhibition (fa ≥0.5) yielded a similar effect when compared with the binary combinations of the tested drugs. When considering results of the CalcuSyn-calculated CI values of the experimental data points, although double combinations of AMY are not synergistically effective, there are examples where it has a synergistic effect with triple combinations (Supplementary Table S5).

Second, according to the DRI values of the experimental data points calculated with the help of CalcuSyn, many combinations of synergistic binary and ternary drugs reached the appropriate DRI values (DRI >1). The results also show favorable dose reduction (DRI >1), as shown in the fa-DRI table in the CalcuSyn report. DRI = 1, DRI >1, and DRI <1 indicate no dose reduction, favorable, and not favorable dose reduction, respectively, for each drug in the combination. To treat malignant diseases such as cancers and AIDS, the concomitant use of drugs is quite common. The main goal of the combination therapy is to provide synergistic effects (CI <1) and reduce the dose of specific toxic drugs (DRI >1) while also relieving the possibility of drug resistance as a result.¹⁸ The DRIs of amygdalin and other drugs were analyzed in both binary and ternary modes in vitro using their IC₅₀ values. As shown in Supplementary Table S6, the DRI values of AMY were much greater than 1 for all combinations on cell lines except for FL. The DRI values of AMY were much smaller than those of all molecules except for MTF and CIS against FL cells, suggesting that cotreatment of AMY is an effective strategy to reduce the dosage and toxicity. In addition, the DRI values of AMY were much higher than those of the other molecules against all cells, implying that combined therapy may reduce both AMY and some other drug doses. However, a detailed examination of Supplementary Table S6 is presented below. At 50% inhibition (fa = 0.5) for MG-63 cells, the combination of AMY and CIS or 5FU or OX or COL or MTZ yielded favorable DRIs ranging from 1.43E+00 to 7.40E+05-fold dose reduction. The combination of AMY and 5FU or COL or CPT yielded favorable DRIs ranging from 1.03E+02 to 2.58E+05-fold dose reduction at fa = 0.5 in Saos2 cells (Supplementary Table S6). At 50% inhibition (fa = 0.5) for SW-1353 cells, the combination of CIS (2.51E+00-fold dose reduction) and CPT (2.97E+02-fold dose reduction) with AMY yielded favorable DRIs (Supplementary Table S6).

Third, in FL cells, Dm values (similar to IC₅₀) of tested binary and ternary compounds were found to range from 5.58E+15 to 3.97E+07 μM and 3.82E+22 to 8.25E+07 μM, respectively (Supplementary Table S4a). However, Dm values (IC₅₀) of tested binary and ternary compounds in other cells were measured between 3.37E+13–5.45E+03 μM and 1.24E+20–2.15E+03 μM, respectively (Supplementary Table S4a). When examining the Dm values for AMY combinations, they were found to be more effective against cancer cells compared with the normal cell (FL) in terms of the cytotoxic activities of these drugs. Hence, findings for combinations of AMY were considered as the second phase of the study for MG63, Saos2, and SW1353 cells. This means that AMY combinations are not toxic to normal cells. AMY and conventional drugs combinations were the most effective and recommended for their cytotoxic activity in the MG63, Saos2, and SW1353 cell lines. Thus, when AMY is administered together with conventional drugs, we believe that effective treatment may be provided at a lower dose of conventional drugs. We concluded that AMY combinations had lower cytotoxicity than conventional drugs. Therefore, combinations of AMY will work for further studies in cancer cells.

The IC₅₀ values of these molecules were also calculated using the four-parameter logistic function. When IC₅₀ values of the compounds were examined, it was observed that these significantly inhibited the proliferation of all tested cell lines, with IC₅₀ values between 5.26 and 397.7 μM (Supplementary Table S4b). However, IC₅₀ values of AMY for MG63 cells, IC₅₀ values of AMY, MTZ, or 5FU for Saos2 cells, and IC₅₀ values of AMY and MTZ for SW1353 cells are larger than 500 μM, implying that they are not effective (Supplementary Table S4b).

DISCUSSION

In chemotherapy, most patients respond well to initial treatment, but in many cases, treatment fails within 3 years. The second-line treatment must begin after the first-line treatment, which may have collapsed due to persistent resistance and is now ineffective. To make the second-line treatment successful and overcome persistent drug resistance, various combination regimens that achieve the same or more substantial effects in different ways have led to a significant improvement in treatment strategy. One of the approaches to increase the second-line therapeutic efficacy is to add natural compounds to sensitize cells to the cytotoxic activity of drugs. Many drugs are in use for treatment of various cancers, and natural phytochemicals such as amygdalin may be used as adjuvant therapy in cancer. For several decades, the antitumor effect of amygdalin has been widely studied. Combination treatment may provide better outcomes and reduce the dosage of conventional drugs required for cytotoxicity. In this study, the interaction between amygdalin and other FDA-approved drugs was experimentally demonstrated. Previous reports have shown that a low concentration of amygdalin could have a slight effect on the cells. However, high concentration of amygdalin can significantly inhibit cell proliferation and induce apoptosis with accompanying cytotoxicity. In the clinic, several cases of cyanide toxicity on ingestion have been reported.¹⁹ Considering the severe toxicity of amygdalin at high dosage, we have chosen a relatively low concentration of amygdalin in combination with some anticancer, antiparasitic, and antigout drugs to study the synergistic inhibitory effect against MG63, Saos2, SW1353, and FL cell proliferation. Although the recent disappointing studies of amygdalin in some tumors did not show the expected outcome, here, significant effects of the combined use of amygdalin and conventional chemotherapeutic agents against MG63, Saos2, and SW1353 cells have been observed.

We have established that amygdalin induces antiproliferative effects in human Saos2 and SW1353 cells as well as displays a prominent adjuvant property to other medicines on MG63 and FL cells, indicating its broad anticancer activity. The adjuvant property of amygdalin may be due to its antioxidant effect that reduces endoplasmic reticulum stress. Amygdalin shows its antioxidant effect by attenuating the effect of alanine transaminase and aspartate transaminase and by reducing malondialdehyde and myeloperoxidase levels.^20
–22 Several studies have shown that amygdalin inhibits renal fibrosis, hyperglycemia, and angiogenesis and relieves symptoms of asthma, bronchitis, and emphysema, as well as exhibits anti-inflammatory and anticancer effects in vitro and in vivo.^23,24 The effects of amygdalin alone and amygdalin combinations with some FDA-approved drugs on these cell lines were investigated for the first time. Previous studies of the effects of amygdalin on other cells have shown different findings. Liczbiński and Bukowska explained that amygdalin exerted multiple effects such as induction of apoptosis and inhibition of adhesion on breast, lung, and bladder cancer cells.²⁵ In conclusion, exposing renal cell carcinoma (Caki-1, KTC-26, and A498) cells to amygdalin inhibits metastatic spread and is associated with downregulation of α5 and α6 integrins. Therefore, amygdalin exerts antitumor activity in vitro.²⁶

In conclusion, the high success rate of drug combinations that cause complete remission in patients whose disease recurs or does not heal provides a driving force to reveal the biological effects of these therapeutic partnerships. The in vitro use of double and triple combination applications presented in this study is of great benefit compared with conventional monodrug applications; however, it is very important to examine the effects on toxicity in in vivo use. An advantage of using drug combinations is that lower concentrations of anticancer drugs can be used because each drug can act through different mechanisms. This study shows that the combination of amygdalin and conventional drugs can increase the cytotoxic effect on MG63, Saos2, and SW1353 cells. Overall, these results show that the combination of amygdalin and traditional chemotherapeutic agents may have significant clinical potential for treatment of bone cancers and deserves more preclinical and clinical studies.

Footnotes

Acknowledgment

The authors are thankful to Dr. Ahmet Karadağ and Dr. Şaban Tekin for their generous assistance.

Authors' Contributions

D.A., K.O., and A.A. participated in the study design and conducted experimental studies and prepared the manuscript. All authors have read and approved the final version of the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Scientific Research Projects of Medeniyet University (Grant No. T-GAP-2017-991).

Supplementary Material

Supplementary Data

Supplementary Figure S1

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

Supplementary Table S7

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Supplementary Material

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