Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy

  • Authors:
    • Heba Emam
    • Alaa Refaat
    • Paras Jawaid
    • Mati Ur Rehman
    • Peng Li
    • Qing‑Li Zhao
    • Takashi Kondo
  • View Affiliations

  • Published online on: May 26, 2017     https://doi.org/10.3892/ol.2017.6256
  • Pages: 1035-1040
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Bufadienolides are constituents of the traditional Chinese medicine Chan Su and are found in toad venom. Cardiovascular side‑effects are one of the limiting factors towards developing bufadienolides as chemotherapeutic agents. Thus, in the present study, low doses of bufalin and cinobufotalin, prominent members of the bufadienolides, were investigated for their cytotoxic activity in combination with hyperthermia (HT) or radiation (Rad) therapy. In addition, the underlying mechanism involved was investigated. A DNA fragmentation assay, viability assay and microscopic observation were primarily used to assess the effect of low doses of the two drugs in human lymphoma U937 cells. Furthermore, the effects of these drugs on the mitochondrial membrane potential (MMP) and apoptotic‑associated protein activation were investigated. HT/bufadienolide‑ and RT/bufadienolide‑treated samples significantly increased the DNA fragmentation percentile and decreased the MMP, as well as increasing the apoptotic features observed microscopically within a relatively short time (6 h) after treatment. The two combinations affected the expression of important apoptotic markers, including caspase‑3 and BH3 interacting domain death agonist. The findings of the current study confirm the additive effect of HT with this group of drugs, directing a novel therapeutic avenue for the clinical use of bufadienolides at lower doses with more restrained cardio toxic side‑effects.

Introduction

Bufalin (B) and cinobufotalin (CB) are two members of the cardiotonic steroids known as the bufadienolides and constituents of the traditional Chinese medicine Chan Su. The drugs are also known to be found in toad venom, which is extracted from the skin secretions of giant toads, including Bufo gargarizans (1). In a previous study, we found that CB was able to significantly induce apoptotic cell death in the human lymphoma U937 cell line at concentrations >0.5 µM (2). In addition, B was demonstrated to significantly induce apoptotic cell death in various cancer cell types at concentrations >0.1 µM (36). The preferential killing of tumor cells by bufadienolides has made them potential candidates for chemotherapeutic research in the last 50 years; however, owing to their structural resemblance to digitalis glycosides, they also act as potent cardiotonic steroids, which increase the contractile force of the heart muscle. This effect is a result of the action of bufadienolides on the Na+/K+-ATPase pump, an essential membrane protein of animal cells, which results in an increase in the cytoplasmic Ca2+ concentration (1). The application of bufadienolides in cancer treatment is, therefore, restricted by their cardiotoxic side-effects.

Previous studies have revealed that hyperthermia (HT) and radiation (Rad) are able to enhance the cell killing effect of cytotoxic drugs, termed thermal- and radio-chemosensitization, either directly through affecting drug activity or indirectly through affecting the cancer cell itself (712). The adjuvant effect of this combination allows for the lowering of the chemotherapeutic dose required, thus lowering the unfavorable side-effects. Overall, the benefits of their concomitant use evidently overweigh the possible aforementioned disadvantages. For this reason, the scope of the present study was to investigate the possibility of lowering the chemotherapeutic dose of bufadienolides in combination with HT or Rad, while maintaining the chemotherapeutic efficacy.

Materials and methods

Drug and drug concentration

CB was purchased from Sigma-Aldrich (Merck KGgA, Darmstadt, Germany), while B was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Stock solutions were prepared using DMSO as a solvent, and further dissolved to make the desired concentrations for experimental use. The minimum toxic doses of CB and B were then determined using DNA fragmentation and cell viability assays, and were identified as 0.2 and 0.1 µM, respectively (data not shown). Accordingly, further experiments were conducted using these concentrations for 6-h incubation periods.

Cell culture

The human lymphoma U937 cell line, obtained from the Health Sciences Research Resource Bank (Japan Health Sciences Foundation, Tokyo, Japan) was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; both Sigma-Aldrich; Merck KGaA). The cell line was maintained at 37°C in a humidified atmosphere with 5% CO2. Cells were harvested when they reached 80% confluence and subcultures of 1×106 cells/ml were used in all assays.

Heat stress exposure

HT is known to cause cell membrane damage and impairment to the ion transport mechanisms (7). Thus, treating the cells with HT prior to B and CB administration diminishes the cytotoxic activity of the drugs that principally target the Na+/K+ pump. The drugs (0.2 µM CB and 0.1 µM B) were therefore administered for 1 h prior to HT treatment. Cells were transferred into test tubes and placed in a hot water bath at 44°C for 20 min. Subsequently, the cells were transferred into plates and incubated for 6 h at 37°C, prior to the required assay.

X-irradiation

U937 cells (1×106 cells/ml) were treated with B and CB in a 6-cm culture dish for 1 h at room temperature prior to Rad. X-irradiation was performed at room temperature using X-ray apparatus (MBR-1520R-3; Hitachi, Ltd., Tokyo, Japan) operating at 150 kV and 20 mA, at a dose rate of 5 Gy/min, 10 Gy total as determined using Fricke dosimetry. Cells were subsequently incubated for 6 h at 37°C with 5% CO2 for further assessment.

Assessment of apoptosis

Morphological examination was performed using Giemsa staining under a light microscope (magnification, ×400), and a quantitative assay of DNA fragmentation was performed according to the modified Sellins and Cohen method (13,14).

Assessment of cell viability

Cell viability was evaluated by WST-1 cell viability assay using a WST-1 Cell Counting kit (Dojindo Molecular Technologies Inc., Kumamoto, Japan) according to the manufacturer's protocol. Briefly, cell suspensions were subjected to drug (CB or B) treatment, HT or Rad, then equal volumes of the cell suspension were seeded into 96-well plate followed by a further incubation period for 6 h at 37°C. Subsequently, cells were subjected to the WST-1 assay. The absorbance was measured at 450 nm using a microplate reader.

Assessment of mitochondrial membrane potential (MMP)

To measure the changes in the MMP, U937 cells were harvested by centrifugation at 350 × g at 4°C for 4 mins and stained with 10 nM tetramethylrhodamine methyl ester (TMRM; Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 15 min at 37°C in PBS containing 1% FBS. The fluorescence of TMRM was then analyzed using flow cytometry (excitation wavelength, 488 nm; emission wavelength, 575 nm) (15).

Western blot analysis of proteins

Western blot analysis was performed as described previously (2). Cells were collected and washed with cold PBS, then lysed using lysis buffer for 20 min at 4°C (1 M Tris-HCl, 5 M NaCl, 1% Nonidet P-40 (v/v), 1% sodium deoxycholate, 0.05% SDS, 1 mM phenylmethylsulfonyl fluoride). Following brief sonication, the lysates were centrifuged at 13,362 × g for 10 min at 4°C and the protein content in the supernatant was measured using a Bio-Rad protein system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total protein (30 µg) was heated at 96°C for 5 min following mixing with 5 µl of SDS-loading buffer. Samples were then subjected to 12% SDS-PAGE (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were then blocked using 1X Tris-buffered saline-Tween 20 with 5% w/v non-fat dry milk for 1 h at room temperature. Western blot analysis was performed using anti-BH3 interacting domain death agonist (Bid,), anti-caspase-3 and anti-heat shock 70-kDa protein (HSP-70; 2002S, 9661S and 4872S, respectively; Cell Signaling Technology, Inc., Danvers, MA, USA) polyclonal antibodies, and anti-β-actin monoclonal antibody (A2228, Sigma-Aldrich; Merck KGgA). The membranes and the primary antibodies (all at 1:1,000 dilution) were incubated overnight with gentle agitation at 4°C. Following their incubation with horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G secondary antibodies for 1 h at room temperature, band signals were visualized on X-ray film using a chemiluminescence EspspCL system (NA934 and NA931, respectively; GE Healthcare Life Sciences, Chalfont, UK) (15).

Statistical analysis

The statistical analysis was performed using JMP software (version 10; SAS Institute, Inc., Cary, NC, USA). Data are presented as the mean ± standard deviation. Statistical significance was evaluated using one-way analysis of variance followed by the Bonferroni post hoc test. All experiments were performed in triplicate. P<0.01 was considered to indicate a statistically significant difference.

Results

Effect of combining HT or Rad on the cytotoxic activity of B and CB

Based on our previous studies, the minimum toxic doses for B and CB in U937 cells were identified as 0.01 and 0.2 µM, respectively (2,5,6). Upon further analysis, administration of B (0.1 µM) and CB (0.2 µM) in combination with HT (44°C for 20 min) or Rad (10 Gy) was demonstrated to significantly increase the DNA fragmentation compared with single-drug treatment groups (P<0.01; Fig. 1A). Treated cells were then examined using Giemsa staining under a light microscope. Untreated cells exhibited intact nuclear structure, whereas B and CB in combination with HT-treated cells showed typical apoptotic morphological changes, as indicated by chromosomal condensation and nuclear fragmentation (Fig. 1B).

Decrease in cell viability

U937 cells were treated as aforementioned, followed by use of the WST-1 cell viability assay. In coherence with the previous assays, B/HT- and CB/HT-treated cells showed a significant decrease in cell viability, reaching 40.0±4.0 and 35.0±3.0%, respectively, following 6 h of incubation (Fig. 2). Similarly, B/Rad- and CB/Rad-treated cells showed a further decrease in cell viability, reaching 20.0±4.0 and 15.0±3.0%, respectively, following 6 h of incubation (Fig. 2).

Induction of MMP

To assess the effect of these combinations on the MMP of U937 cells, treated cells were assessed for MMP decline using TMRM staining. The results revealed significant reductions in MMP in the combination-treatment groups compared with treatment with the drugs alone, as shown in Fig. 3.

Effect on apoptosis-associated protein expression

Caspases serve an important role in the apoptotic signaling pathway and are considered as the main executioners of cell death (16). To examine the involvement of the caspases in the effect of HT or Rad on the two drugs, western blot analysis was performed using caspase-3 antibody following 6 h of treatment of U937 cells using the treatment combinations. Given that the activation of a specific caspase results in the reduction of its pro-enzyme form, it was demonstrated that the protein levels of procaspase-3 were markedly downregulated in the HT- or Rad-treated groups (Fig. 4). Simultaneously, the cleaved form of caspase-3 was also markedly detectable in the groups treated in combination with HT or Rad. Expression of activated pro-apoptotic proteins, including Bid, were also demonstrated to be markedly decreased with regard to cytosolic levels, as shown in Fig. 4. However, HSP70 expression was upregulated in the HT-treated groups.

Discussion

Cardiac glycosides (CG) belong to the most commonly used group of drugs in medicine. The two main subgroups of CG are the cardenolides, where ouabin and its derivative digoxin are the most typical examples, and the bufadienolides, which the present study was focused upon. There are ~250 substances in the bufadienolide group, including B and CB (17). The bufadienolides have been previously investigated in several studies for their cytotoxic activity, as well as the underlying signaling pathway involved (36). Bufadienolides act primarily through inhibiting the transport enzyme Na+/K+-adenosine triphosphatase (18), thus acting as their signal transducer. Conversely, despite the cytotoxic nature of bufadienolides, a previous study reported their use in cardiovascular and kidney diseases (19). Introducing the bufadienolides as chemotherapeutic agents also depends on the extent of their cardiovascular side-effects. However, their cell type specificity towards cancer cells and the use of specific bufadienolides at low doses and for long periods without severe side-effects have been reported (20,21). Thus, in the present study, the use of B and CB was investigated at the lowest cytotoxic dose in combination with HT or Rad in order to attain a significant cytotoxicity with limited possible side-effects.

HT efficacy is not sufficient to replace any of the therapeutic modalities when used alone, yet it has shown significant results in enhancing the cell killing effect of other cytotoxic drugs. HT induces several changes in the cellular physiology, including at the level of the cell membrane of the cancer cell and on a nucleic acid level (7). Generally, HT cannot cause severe damage by itself, but instead, independently hinders the repair of cell damage (22,23). Furthermore, HT affects the fluidity and stability of cellular membranes, and hinders the function of the transmembrane transport proteins and cell surface receptors (2428). In addition, X-irradiation is known to induce apoptosis by acting directly on the plasma membrane and nuclear DNA, or both (14). Simultaneously, mitochondrial-dependent generation of reactive oxygen species serves an essential role in G2/M arrest and X-irradiation-induced apoptosis (29,30). Accordingly, the present study reported promising results when B and CB were administered 1 h prior to HT or Rad treatment avoiding its destructive effect on the cell membrane, which is the primary target of B and CB.

In all the assays in the current study, B was identified to be generally more cytotoxic when compared with CB, and HT significantly enhanced the cytotoxic effect of low doses of the two drugs. Notably, HT was able to potentiate the cytotoxic activity of CB to be almost equal to that of B alone. The apoptotic nature of the drugs appeared to be unhindered yet augmented. Firstly, using Giemsa staining, the presence of apoptotic-like bodies in all treatment groups was demonstrated and was more evident in the CB/HT and B/HT combinations compared with the groups with drug treatment alone. To assess the viability of the cells, a WST-1 cell viability assay was used. The results demonstrated that ~50% of the cells died within 6 h in the CB/HT and B/HT combination groups vs. 20–30% of cells when the drugs were used alone. Additionally, the DNA fragmentation assay revealed a significant increase in the DNA fragments in the combination groups. Furthermore, the effects of the combinations were greater when assessing the MMP, since there was doubling in the MMP percentile loss in the two drugs. MMP is the preliminary step towards the initiation of programmed cell death (31,32). This eventually leads to the activation of caspase-3, followed by DNA fragmentation and cell death (33,34). HT and Rad enhanced the expression of caspase-3 and Bid proteins when combined with the drugs. However, the upregulation of HSP70, acknowledged as an anti-apoptotic protein, remains a question to be assessed. Nevertheless, few previous studies have demonstrated the apoptotic nature of the HSP70 protein. This could stand as an explanation for the lack of a synergistic effect of this combination (35).

In conclusion, HT and Rad, in combinations with low doses of B and CB appear to enhance the apoptotic cell death of U937 cells through the intrinsic apoptotic signaling pathway. Notably, when combined with CB, chemotherapeutic effects were demonstrated equivalent to the relatively more toxic B used alone. The present study has provided a direction for the introduction of B and CB into the chemotherapeutic field, ensuring similar efficacy while reducing the anticipated side-effects of high doses.

References

1 

Xu Y, Liu X, Schwarz S, Hu L, Guo D, Gu Q and Schwarz W: Inhibitory efficacy of bufadienolides on Na+, K+-pump activity versus cell proliferation. Biochem Biophys Rep. 6:158–164. 2016.

2 

Emam H, Zhao QL, Furusawa Y, Refaat A, Ahmed K, Kadowaki M and Kondo T: Apoptotic cell death by the novel natural compound, cinobufotalin. Chem Biol Interact. 199:154–160. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Yin PH, Liu X, Qiu YY, Cai JF, Qin JM, Zhu HR and Li Q: Anti-tumor activity and apoptosis-regulation mechanisms of bufalin in various cancers: New hope for cancer patients. Asian Pac J Cancer Prev. 13:5339–5343. 2012. View Article : Google Scholar : PubMed/NCBI

4 

Takai N, Kira N, Ishii T, Yoshida T, Nishida M, Nishida Y, Nasu K and Narahara H: Bufalin, a traditional oriental medicine, induces apoptosis in human cancer cells. Asian Pac J Cancer Prev. 13:399–402. 2012. View Article : Google Scholar : PubMed/NCBI

5 

Watabe M, Ito K, Masuda Y, Nakajo S and Nakaya K: Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells. Oncogene. 16:779–787. 1998. View Article : Google Scholar : PubMed/NCBI

6 

Watabe M, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y and Nakaya K: The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells. J Biol Chem. 271:14067–14072. 1996. View Article : Google Scholar : PubMed/NCBI

7 

Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T, Felix R and Riess H: The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol. 43:33–56. 2002. View Article : Google Scholar : PubMed/NCBI

8 

Engin K, Tupchong L, Moylan DJ, Alexander GA, Waterman FM, Komarnicky L, Nerlinger RE and Leeper DB: Radomized trial of one versus two adjuvant hyperthermia treatments per week in patients with superficial tumors. Int J Hyperthermia. 9:327–340. 1993. View Article : Google Scholar : PubMed/NCBI

9 

Urano M, Kuroda M and Nishimura Y: For the clinical application of themochemotherapy given at mild temperatures. Int J Hyperthermia. 15:79–107. 1999. View Article : Google Scholar : PubMed/NCBI

10 

Falk MH and Issels RD: Hyperthermia in oncology. Int J Hyperthermia. 17:1–18. 2001. View Article : Google Scholar : PubMed/NCBI

11 

Dewey WC, Thrall D and Gilette EL: Hyperthermia and radiation-a selective thermal effect on chronically hypoxic tumor cells in vivo. Int J Radiat Oncol Biol Phys. 2:99–103. 1977. View Article : Google Scholar : PubMed/NCBI

12 

Dewey WC, Hopwood LE, Sapareto SA and Gerweck LE: Cellular responses to combinations of hyperthermia and radiation. Radiology. 123:463–474. 1977. View Article : Google Scholar : PubMed/NCBI

13 

Sellins KS and Cohen JJ: Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol. 139:3199–3206. 1987.PubMed/NCBI

14 

Hopcia KL, McCarey YL, Sylvester FC and Held KD: Radiation- induced apoptosis in HL60 cells: Oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose. Radiat Res. 145:315–323. 1996. View Article : Google Scholar : PubMed/NCBI

15 

Zhao QL, Fujiwara Y and Kondo T: Mechanism of cell death induction by nitroxide and hyperthermia. Free Radic Biol Med. 40:1131–1143. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Utz PJ and Anderson P: Life and death decisions: Regulation of apoptosis by proteolysis of signaling molecules. Cell Death Differ. 7:589–602. 2000. View Article : Google Scholar : PubMed/NCBI

17 

Krenn L and Kopp B: Bufadienolides from animal and plant sources. Phytochemistry. 48:1–29. 1998. View Article : Google Scholar : PubMed/NCBI

18 

Schatzmann HJ: The role of Na+ and K+ in the ouabain-inhibition of the Na+ K+-activated membrane adenosine triphosphatase. Biochim Biophys Acta. 94:89–96. 1965. View Article : Google Scholar : PubMed/NCBI

19 

Puschett JB, Agunanne E and Uddin MN: Emerging role of the bufadienolides in cardiovascular and kidney diseases. Am J Kidney Dis. 56:359–370. 2010. View Article : Google Scholar : PubMed/NCBI

20 

Jing Y, Ohizumi H, Kawazoe N, Hashimoto S, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y and Nakaya K: Selective inhibitory effect of bufalin on growth of human tumor cells in vitro: Association with the induction of apoptosis in leukemia HL-60 cells. Jpn J Cancer Res. 85:645–651. 1994. View Article : Google Scholar : PubMed/NCBI

21 

Panesar NS: Bufalin and unidentified substance(s) in traditional Chinese medicine cross-react in commercial digoxin assay. Clin Chem. 38:2155–20156. 1992.PubMed/NCBI

22 

Dahm-Daphi J, Brammer I and Dikomey E: Heat effects on the repair of DNA double-strand breaks in CHO cells. Int J Radiat Biol. 72:171–179. 1997. View Article : Google Scholar : PubMed/NCBI

23 

Dikomey E and Franske J: Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells. Int J Radiat Biol. 61:221–233. 1992. View Article : Google Scholar : PubMed/NCBI

24 

Calderwood SK and Hahn GM: Thermal sensitivity and resistance of insulin-receptor binding. Biochim Biophys Acta. 756:1–8. 1983. View Article : Google Scholar : PubMed/NCBI

25 

Stevenson MA, Minton KW and Hahn GM: Survival and concanavalin- A-induced capping in CHO fibroblasts after exposure to hyperthermia, ethanol, and X irradiation. Radiat Res. 86:467–478. 1981. View Article : Google Scholar : PubMed/NCBI

26 

Coss RA and Linnemanns WA: The effects of hyperthermia on the cytoskeleton: A review. Int J Hyperthermia. 12:173–196. 1996. View Article : Google Scholar : PubMed/NCBI

27 

Konings AW and Ruifrok AC: Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells. Radiat Res. 102:86–98. 1985. View Article : Google Scholar : PubMed/NCBI

28 

Majda JA, Gerner EW, Vanlandingham B, Gehlsen KR and Cress AE: Heat shock-induced shedding of cell surface integrins in A549 human lung tumor cells in culture. Exp Cell Res. 210:46–51. 1994. View Article : Google Scholar : PubMed/NCBI

29 

Miyata H, Doki Y, Yamamoto H, Kishi K, Takemoto H, Fujiwara Y, Yasuda T, Yano M, Inoue M, Shiozaki H, et al: Overexpression of CDC25B overrides radiation-induced G2-M arrest and results in increased apoptosis in esophageal cancer cells. Cancer Res. 61:3188–3193. 2001.PubMed/NCBI

30 

Corbiere C, Liagre B, Terro F and Beneytout JL: Induction of antiproliferative effect by diosgenin through activation of p53, release of apoptosis-inducing factor (AIF) and modulation of caspase-3 activity in different human cancer cells. Cell Res. 14:188–196. 2004. View Article : Google Scholar : PubMed/NCBI

31 

Gottlieb E, Armour SM, Harris MH and Thompson CB: Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ. 10:709–717. 2003. View Article : Google Scholar : PubMed/NCBI

32 

Gottlieb RA: Mitochondria: Execution central. FEBS Lett. 482:6–12. 2000. View Article : Google Scholar : PubMed/NCBI

33 

Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B and Kroemer G: Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med. 182:367–377. 1995. View Article : Google Scholar : PubMed/NCBI

34 

Liu X, Kim CN, Yang J, Jemmerson R and Wang X: Induction of apoptotic program in cell-free extract: Requirements for dATP and cytochrome C. Cell. 86:147–157. 1996. View Article : Google Scholar : PubMed/NCBI

35 

Ran R, Lu A, Zhang L, Tang Y, Zhu H, Xu H, Feng Y, Han C, Zhou G, Rigby AC and Sharp FR: Hsp70 promotes TNF-mediated apoptosis by binding IKK gamma and impairing NF-kappa B survival signaling. Genes Dev. 18:1466–1481. 2004. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

July-2017
Volume 14 Issue 1

Print ISSN: 1792-1074
Online ISSN:1792-1082

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Emam H, Refaat A, Jawaid P, Ur Rehman M, Li P, Zhao QL and Kondo T: Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy. Oncol Lett 14: 1035-1040, 2017
APA
Emam, H., Refaat, A., Jawaid, P., Ur Rehman, M., Li, P., Zhao, Q., & Kondo, T. (2017). Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy. Oncology Letters, 14, 1035-1040. https://doi.org/10.3892/ol.2017.6256
MLA
Emam, H., Refaat, A., Jawaid, P., Ur Rehman, M., Li, P., Zhao, Q., Kondo, T."Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy". Oncology Letters 14.1 (2017): 1035-1040.
Chicago
Emam, H., Refaat, A., Jawaid, P., Ur Rehman, M., Li, P., Zhao, Q., Kondo, T."Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy". Oncology Letters 14, no. 1 (2017): 1035-1040. https://doi.org/10.3892/ol.2017.6256