- Email: [email protected]
Crocin improves the proliferation and cytotoxic function of T cells in children with acute lymphoblastic leukemia
Biomedicine & Pharmacotherapy 99 (2018) 96–100
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Crocin improves the proliferation and cytotoxic function of T cells in children with acute lymphoblastic leukemia Kunpeng Zhang1, Lingzhen Wang1, Shaoyong Si, Yan Sun, Wenting Pei, Yan Ming, Lirong Sun
T ⁎
Department of Pediatrics Hematology, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Crocin Ara-C T cells DNA damage Acute lymphoblastic leukemia
Objective: Immunotherapy is important to improve the survival of children with acute lymphoblastic leukemia (ALL). This study aimed to assess the effects of crocin on the proliferation and function of T cells isolated from children with ALL. Methods: The mononuclear cells were isolated from peripheral blood of children with ALL and then treated with different final concentrations of crocin. The levels of different cytokines secreted by T cells and the ratio of CD4 and CD8 were measured. Tail DNA% (TDNA), Tail moment (TM), Tail length (TL) and sister chromatid exchange (SCE) were detected to assess DNA damage of T cells. Results: Crocin significantly promoted T cell proliferation and the secretion of IL-2 and IL-4 in a concentration dependent manner. In addition, crocin increased CD4/CD8 ratio of T subset. Crocin itself caused no significant damage to T cells but reduced DNA damage in T cells treated with Ara-C. Conclusions: Crocin could improve the proliferation and cytotoxic function of T cells, and reduce DNA damage caused by Ara-C.
1. Introduction
2. Materials and methods
Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of children. The survival rate has increased for children with acute leukemia with the development of intensive chemotherapy, but 30–50% children with leukemia still relapse [1]. The minimal residual disease (MRD) of leukemia induces leukemia relapse, and immunotherapy is one important approach for improving the survival rate of children with leukemia [2]. Crocin is a main water-soluble carotenoid of the saffron extract, which belongs to perential stemless herb of the large Inridaceae family. Saffron extracts inhibited the proliferation of human acute lymphoblastic T-cell human leukemia [3]. Growing evidence has shown that crocin could significantly inhibit the proliferation of cancer cells [4]. Our previous study reported that crocin inhibited the proliferation of HL-60 cells [5]. However, the effect of crocin on immune cells has not been evaluated in detail. In the present study, we performed a series of experiments to assess the effects of crocin on the lymphocytes from children with acute leukemia for complete remission.
2.1. Preparation of T lymphocytes
⁎
1
Peripheral blood mononuclear cells (PBMCs) of ALL children who had complete remission for at least 6 months were isolated by Ficoll’s density gradient centrifugation method, and were seeded into 24 well flat-bottom culture plates. All blood samples were obtained with informed consent from their parents, which were approved by the Institute Ethics Committee. After culture for 3 h, non-adherent lymphocytes were taken and further cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified incubator of 5% CO2 at 37 ℃. 2.2. Cell proliferation assay Crocin (Sigma, CAS Number 42553-65-1) was diluted in phosphatebuffered saline for the appropriate concentration. T lymphocytes were divided into different groups and treated with crocin (final concentration 0.625–2.5 mg/ml) for 72 h. Cells were treated with phytohe-
Corresponding author. E-mail address: [email protected] (L. Sun). These are are co-first authors.
https://doi.org/10.1016/j.biopha.2018.01.042 Received 5 December 2017; Received in revised form 28 December 2017; Accepted 5 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
Fig. 1. Crocin promoted the proliferation of T cells. (A)The morphology of T cells under microscope. The lymphocytes in crocin group showed more number, presenting cluster-shape. (B) The proliferation index of T cells gradually increased with the increase of crocin concentration (n = 5).*p < 0.05, compared with control group and PHA group.
Fig. 2. Crocin increased the cytokines secreted by T cells. (A) ELISA assay of IL-2 level secreted by T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ml) (n = 5). (B) ELISA assay of IL-4 level secreted by T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ml) (n = 5). *p < 0.05, compared with PHA group and control group.
2.4. ELISA
magglutinin (PHA) as positive control. The cell proliferation was examined by MTT assay kit (Sigma).
The supernatants of T lymphocytes were collected and the concentrations of cytokines IL-2 and IL-4 were examined by using ELISA kits (R&D systems, Minneapolis, MN, USA) following the manufacturer’s instructions.
2.3. Flow cytometry T lymphocytes were washed and stained with fluorescence conjugated monoclonal antibodies for CD4 and CD8. Next the cells were fixed in 1% formaldehyde and ≥10,000 cells per sample were immediately analyzed on a flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed by using FlowJo software (FlowJo, Ashland, OR, USA).
2.5. Comet assay T lymphocytes were treated with different concentration of crocin for 72 h. Cells treated with Ara-C (Cytarabine) or Ascorbic acid (Zel C) were included as control groups. The length of the comet tail in μm 97
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
3.2. Crocin increased the secretion of IL-2 and IL-4 by T cells The supernatant from T cells treated with crocin was used to assess the level of IL-2 and IL-4. ELISA showed that crocin increased the secretion of IL-2 and IL-4 in a dose dependent manner, and the concentrations of secreted cytokines were the highest when the concentration of crocin was 2.5 mg/ml (Fig. 2). 3.3. Crocin improved the ratio of CD4/CD8 of T cells Flow cytometry showed that the ratio of CD4/CD8 of T cells increased with the increase of crocin concentration. CD4/CD8 ratio was the highest when the concentration of crocin was 2.5 mg/ml (Fig. 3).
Fig. 3. The CD4/CD8 ratio in T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ ml) (n = 5). *p < 0.05, compared with PHA group and control group.
3.4. Crocin caused no significant DNA damage to T cells (TL), the percentage of DNA damage in the tail (TDNA) and tail moment (TM) were recorded with single-cell gel electrophoresis assay. The number of sister chromatid exchange (SCE) with sister chromatid differentiation was counted.
As shown in Fig. 4, there was no significant difference in TDNA of T cells treated with crocin from 0.625 mg/ml to 2.5mg/ml and Zel C group, all of which were lower than that in cells treated with Ara-c (12.67 ± 3.36%). TM in Ara-c group was 2.41 ± 0.75, significantly higher than that in crocin group and Zel C group, while TL in crocin group was lower than that in Ara-c group. SCE imaging showed that there was tailing phenomenon in Ara-c group but not in crocin group.
3. Results 3.1. Crocin promoted T cell proliferation
3.5. Crocin improved DNA repair of T cells MTT assay showed that different concentrations (from 0.625 mg/ml to 2.5 mg/ml) of crocin significantly promoted proliferation of T cells and the proliferation index gradually increased with the increase of crocin concentration (Fig. 1).
Next we treated T cells with both crocin and Ara-c and found that TDNA, TM and TL were significantly lower in these cells compared to cells treated with Ara-c alone (Fig. 5). These data indicated that crocin improved DNA repair of T cells.
Fig. 4. Comet assay of DNA damage in T cells. (A) TDNA in each group. (B) TM in each group. (C) Tail length in each group. (D) SCE images of T cells treated with 2.5 mg/ml crocin or Ara-c for 72 h. *p < 0.05, compared with control group and crocin group.
98
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
Fig. 5. Crocin protected DNA damage by Ara-c in T cells. (A) TDNA in each group. (B) TM in each group. (C) Tail length in each group. (D) SCE in each group. *p < 0.05, compared with control group and crocin group.
4. Discussion
damage, similar to the mechanism by which zel C is involved in the repair of genotoxicity [14]. In summary, our study found that crocin caused no DNA damage and reduced Ara-c-induced DNA damage. In addition, crocin promoted T cells proliferation and cytokine secretion. Therefore, crocin is expected to become a new type of anti-tumor drug.
Recently, great attention has been focused on the use of natural compounds as novel agents for leukemia therapy [6,7]. Our previous study showed that crocin could inhibit tumor growth in mice with leukemia [5]. In this study, we assessed the effect of crocin on T cells isolated from ALL patients. Crocin could promote the proliferation of T cells, and increase the cytokine levels secreted by T cells. Moreover, pretreatment of crocin could suppress Ara-c induced DNA damage in a dose-dependent manner. These results are consistent with those reported by Premkumar et al. [8]. It is known that antioxidants could attenuate chemically induced carcinogenesis, showing chemoprotective effect [9]. DNA damage and repair is an important field of genetic toxicology. So far, the most sensitive endpoint of DNA damage is to detect DNA fragmentation [10]. Ara-c is one of the most commonly used chemotherapeutic drugs for acute leukemia. Bullock et al. found that Ara-c inhibited DNA repair-related DNA polymerase to promote leukemia cell apoptosis [11]. Zel C is one kind of reducing agents without genetic toxic effects, and could reduce mycotoxin caused by DNA damage through its antioxidant activity [12]. In our study, SCGE and SCD experiments showed that there was no considerable DNA damage in crocin group and pretreatment of crocin could promote the repair of DNA damage induced by Ara-c. Crocin is the main active ingredient of saffron. Nair et al. found that saffron could increase the level of intracellular reduced glutathione and its related enzymes, suggesting its antioxidant biological activity [13]. Addition studies showed that saffron extract and its active ingredient crocin had antioxidant biological activity [14,15]. We hypothesized that crocin exhibited antioxidant biological activity to reduce DNA
Conflict of interest No References [1] M.C. Ethier, E. Blanco, T. Lehrnbecher, L. Sung, Lack of clarity in the definition of treatment-related mortality: pediatric acute leukemia and adult acute promyelocytic leukemia as examples, Blood 118 (2011) 5080–5083. [2] D.O. Acheampong, C.K. Adokoh, D.B. Asante, E.A. Asiamah, P.A. Barnie, D.O.M. Bonsu, F. Kyei, Immunotherapy for acute myeloid leukemia (AML): a potent alternative therapy, Biomed. Pharmacother. 97 (2017) 225–232. [3] H. Makhlouf, M. Diab-Assaf, M. Alghabsha, M. Tannoury, R. Chahine, A.M. Saab, In vitro antiproliferative activity of saffron extracts against human acute lymphoblastic T-cell human leukemia, Indian J. Trad. Knowl. 15 (2016) 16–21. [4] H. Bakshi, S. Sam, R. Rozati, P. Sultan, T. Islam, B. Rathore, Z. Lone, M. Sharma, J. Triphati, R.C. Saxena, DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from Kashmiri Saffron in a human pancreatic cancer cell line, Asian Pac, J. Cancer Prev. 11 (2010) 675–679. [5] Y. Sun, H.J. Xu, Y.X. Zhao, L.Z. Wang, L.R. Sun, Z. Wang, X.F. Sun, Crocin exhibits antitumor effects on human Leukemia HL-60 cells in vitro and in vivo, Evid. Based Complement. Altern. Med. 2013 (2013) 690164. [6] S. Ben Jannet, N. Hymery, S. Bourgou, A. Jdey, M. Lachaal, C. Magné, R. Ksouri, Antioxidant and selective anticancer activities of two Euphorbia species in human acute myeloid leukemia, Biomed. Pharmacother. 90 (2017) 375–385. [7] C.S. Lima, D.G. Spindola, A. Bechara, D.M. Garcia, C. Palmeira-Dos-Santos, J. Peixoto-daSilva, A.G. Erustes, L.F.G. Michelin, G.J.S. Pereira, S.S. Smaili, E. Paredes-Gamero, A.K. Calgarotto, C.R. Oliveira, C. Bincoletto, Cafestol, a diterpene molecule found in coffee, induces leukemia cell death, Biomed. Pharmacother. 92 (2017) 1045–1054.
99
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
damage assessed by the cytogenetic tests, Toxicol. Ind. Health 28 (2012) 648–654. [13] S.C. Nair, M.J. Salomi, C.D. Varghese, B. Panikkar, K.R. Panikkar, Effect of saffron on thymocyte proliferation, intracellular glutathione levels and its antitumor activity, Biofactors 4 (1992) 51–54. [14] H. Hosseinzadeh, F. Shamsaie, S. Mehri, Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal, Pharmacogn. Mag. 5 (2009) 419–424. [15] M.R. Salahshoor, M. Khazaei, C. Jalili, M. Keivan, Crocin improves damage induced by nicotine on A number of reproductive parameters in male mice, Int. J. Fertil. Steril. 10 (2016) 71–78.
[8] K. Premkumar, C. Thirunavukkarasu, S.K. Abraham, S.T. Santhiya, A. Ramesh, Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice, Hum. Exp. Toxicol. 25 (2006) 79–84. [9] N. Khan, F. Afaq, H. Mukhtar, Cancer chemoprevention through dietary antioxidants: progress and promise, Antioxid. Redox Signal. 10 (2008) 475–510. [10] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantization of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1998) 184–191. [11] G. Bullock, S. Ray, J.C. Reed, S. Krajewski, A.M. Ibrado, Y. Huang, K. Bhalla, Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL60 cells possessing disparate levels of Bcl-2 protein, Leukemia 10 (1996) 1731–1740. [12] H. Türkez, E. Aydin, The protective role of ascorbic acid on imazalil-induced genetic
100
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Crocin improves the proliferation and cytotoxic function of T cells in children with acute lymphoblastic leukemia Kunpeng Zhang1, Lingzhen Wang1, Shaoyong Si, Yan Sun, Wenting Pei, Yan Ming, Lirong Sun
T ⁎
Department of Pediatrics Hematology, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Crocin Ara-C T cells DNA damage Acute lymphoblastic leukemia
Objective: Immunotherapy is important to improve the survival of children with acute lymphoblastic leukemia (ALL). This study aimed to assess the effects of crocin on the proliferation and function of T cells isolated from children with ALL. Methods: The mononuclear cells were isolated from peripheral blood of children with ALL and then treated with different final concentrations of crocin. The levels of different cytokines secreted by T cells and the ratio of CD4 and CD8 were measured. Tail DNA% (TDNA), Tail moment (TM), Tail length (TL) and sister chromatid exchange (SCE) were detected to assess DNA damage of T cells. Results: Crocin significantly promoted T cell proliferation and the secretion of IL-2 and IL-4 in a concentration dependent manner. In addition, crocin increased CD4/CD8 ratio of T subset. Crocin itself caused no significant damage to T cells but reduced DNA damage in T cells treated with Ara-C. Conclusions: Crocin could improve the proliferation and cytotoxic function of T cells, and reduce DNA damage caused by Ara-C.
1. Introduction
2. Materials and methods
Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of children. The survival rate has increased for children with acute leukemia with the development of intensive chemotherapy, but 30–50% children with leukemia still relapse [1]. The minimal residual disease (MRD) of leukemia induces leukemia relapse, and immunotherapy is one important approach for improving the survival rate of children with leukemia [2]. Crocin is a main water-soluble carotenoid of the saffron extract, which belongs to perential stemless herb of the large Inridaceae family. Saffron extracts inhibited the proliferation of human acute lymphoblastic T-cell human leukemia [3]. Growing evidence has shown that crocin could significantly inhibit the proliferation of cancer cells [4]. Our previous study reported that crocin inhibited the proliferation of HL-60 cells [5]. However, the effect of crocin on immune cells has not been evaluated in detail. In the present study, we performed a series of experiments to assess the effects of crocin on the lymphocytes from children with acute leukemia for complete remission.
2.1. Preparation of T lymphocytes
⁎
1
Peripheral blood mononuclear cells (PBMCs) of ALL children who had complete remission for at least 6 months were isolated by Ficoll’s density gradient centrifugation method, and were seeded into 24 well flat-bottom culture plates. All blood samples were obtained with informed consent from their parents, which were approved by the Institute Ethics Committee. After culture for 3 h, non-adherent lymphocytes were taken and further cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified incubator of 5% CO2 at 37 ℃. 2.2. Cell proliferation assay Crocin (Sigma, CAS Number 42553-65-1) was diluted in phosphatebuffered saline for the appropriate concentration. T lymphocytes were divided into different groups and treated with crocin (final concentration 0.625–2.5 mg/ml) for 72 h. Cells were treated with phytohe-
Corresponding author. E-mail address: [email protected] (L. Sun). These are are co-first authors.
https://doi.org/10.1016/j.biopha.2018.01.042 Received 5 December 2017; Received in revised form 28 December 2017; Accepted 5 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
Fig. 1. Crocin promoted the proliferation of T cells. (A)The morphology of T cells under microscope. The lymphocytes in crocin group showed more number, presenting cluster-shape. (B) The proliferation index of T cells gradually increased with the increase of crocin concentration (n = 5).*p < 0.05, compared with control group and PHA group.
Fig. 2. Crocin increased the cytokines secreted by T cells. (A) ELISA assay of IL-2 level secreted by T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ml) (n = 5). (B) ELISA assay of IL-4 level secreted by T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ml) (n = 5). *p < 0.05, compared with PHA group and control group.
2.4. ELISA
magglutinin (PHA) as positive control. The cell proliferation was examined by MTT assay kit (Sigma).
The supernatants of T lymphocytes were collected and the concentrations of cytokines IL-2 and IL-4 were examined by using ELISA kits (R&D systems, Minneapolis, MN, USA) following the manufacturer’s instructions.
2.3. Flow cytometry T lymphocytes were washed and stained with fluorescence conjugated monoclonal antibodies for CD4 and CD8. Next the cells were fixed in 1% formaldehyde and ≥10,000 cells per sample were immediately analyzed on a flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed by using FlowJo software (FlowJo, Ashland, OR, USA).
2.5. Comet assay T lymphocytes were treated with different concentration of crocin for 72 h. Cells treated with Ara-C (Cytarabine) or Ascorbic acid (Zel C) were included as control groups. The length of the comet tail in μm 97
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
3.2. Crocin increased the secretion of IL-2 and IL-4 by T cells The supernatant from T cells treated with crocin was used to assess the level of IL-2 and IL-4. ELISA showed that crocin increased the secretion of IL-2 and IL-4 in a dose dependent manner, and the concentrations of secreted cytokines were the highest when the concentration of crocin was 2.5 mg/ml (Fig. 2). 3.3. Crocin improved the ratio of CD4/CD8 of T cells Flow cytometry showed that the ratio of CD4/CD8 of T cells increased with the increase of crocin concentration. CD4/CD8 ratio was the highest when the concentration of crocin was 2.5 mg/ml (Fig. 3).
Fig. 3. The CD4/CD8 ratio in T cells treated with PHA or crocin (0.625 mg/ml–2.5 mg/ ml) (n = 5). *p < 0.05, compared with PHA group and control group.
3.4. Crocin caused no significant DNA damage to T cells (TL), the percentage of DNA damage in the tail (TDNA) and tail moment (TM) were recorded with single-cell gel electrophoresis assay. The number of sister chromatid exchange (SCE) with sister chromatid differentiation was counted.
As shown in Fig. 4, there was no significant difference in TDNA of T cells treated with crocin from 0.625 mg/ml to 2.5mg/ml and Zel C group, all of which were lower than that in cells treated with Ara-c (12.67 ± 3.36%). TM in Ara-c group was 2.41 ± 0.75, significantly higher than that in crocin group and Zel C group, while TL in crocin group was lower than that in Ara-c group. SCE imaging showed that there was tailing phenomenon in Ara-c group but not in crocin group.
3. Results 3.1. Crocin promoted T cell proliferation
3.5. Crocin improved DNA repair of T cells MTT assay showed that different concentrations (from 0.625 mg/ml to 2.5 mg/ml) of crocin significantly promoted proliferation of T cells and the proliferation index gradually increased with the increase of crocin concentration (Fig. 1).
Next we treated T cells with both crocin and Ara-c and found that TDNA, TM and TL were significantly lower in these cells compared to cells treated with Ara-c alone (Fig. 5). These data indicated that crocin improved DNA repair of T cells.
Fig. 4. Comet assay of DNA damage in T cells. (A) TDNA in each group. (B) TM in each group. (C) Tail length in each group. (D) SCE images of T cells treated with 2.5 mg/ml crocin or Ara-c for 72 h. *p < 0.05, compared with control group and crocin group.
98
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
Fig. 5. Crocin protected DNA damage by Ara-c in T cells. (A) TDNA in each group. (B) TM in each group. (C) Tail length in each group. (D) SCE in each group. *p < 0.05, compared with control group and crocin group.
4. Discussion
damage, similar to the mechanism by which zel C is involved in the repair of genotoxicity [14]. In summary, our study found that crocin caused no DNA damage and reduced Ara-c-induced DNA damage. In addition, crocin promoted T cells proliferation and cytokine secretion. Therefore, crocin is expected to become a new type of anti-tumor drug.
Recently, great attention has been focused on the use of natural compounds as novel agents for leukemia therapy [6,7]. Our previous study showed that crocin could inhibit tumor growth in mice with leukemia [5]. In this study, we assessed the effect of crocin on T cells isolated from ALL patients. Crocin could promote the proliferation of T cells, and increase the cytokine levels secreted by T cells. Moreover, pretreatment of crocin could suppress Ara-c induced DNA damage in a dose-dependent manner. These results are consistent with those reported by Premkumar et al. [8]. It is known that antioxidants could attenuate chemically induced carcinogenesis, showing chemoprotective effect [9]. DNA damage and repair is an important field of genetic toxicology. So far, the most sensitive endpoint of DNA damage is to detect DNA fragmentation [10]. Ara-c is one of the most commonly used chemotherapeutic drugs for acute leukemia. Bullock et al. found that Ara-c inhibited DNA repair-related DNA polymerase to promote leukemia cell apoptosis [11]. Zel C is one kind of reducing agents without genetic toxic effects, and could reduce mycotoxin caused by DNA damage through its antioxidant activity [12]. In our study, SCGE and SCD experiments showed that there was no considerable DNA damage in crocin group and pretreatment of crocin could promote the repair of DNA damage induced by Ara-c. Crocin is the main active ingredient of saffron. Nair et al. found that saffron could increase the level of intracellular reduced glutathione and its related enzymes, suggesting its antioxidant biological activity [13]. Addition studies showed that saffron extract and its active ingredient crocin had antioxidant biological activity [14,15]. We hypothesized that crocin exhibited antioxidant biological activity to reduce DNA
Conflict of interest No References [1] M.C. Ethier, E. Blanco, T. Lehrnbecher, L. Sung, Lack of clarity in the definition of treatment-related mortality: pediatric acute leukemia and adult acute promyelocytic leukemia as examples, Blood 118 (2011) 5080–5083. [2] D.O. Acheampong, C.K. Adokoh, D.B. Asante, E.A. Asiamah, P.A. Barnie, D.O.M. Bonsu, F. Kyei, Immunotherapy for acute myeloid leukemia (AML): a potent alternative therapy, Biomed. Pharmacother. 97 (2017) 225–232. [3] H. Makhlouf, M. Diab-Assaf, M. Alghabsha, M. Tannoury, R. Chahine, A.M. Saab, In vitro antiproliferative activity of saffron extracts against human acute lymphoblastic T-cell human leukemia, Indian J. Trad. Knowl. 15 (2016) 16–21. [4] H. Bakshi, S. Sam, R. Rozati, P. Sultan, T. Islam, B. Rathore, Z. Lone, M. Sharma, J. Triphati, R.C. Saxena, DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from Kashmiri Saffron in a human pancreatic cancer cell line, Asian Pac, J. Cancer Prev. 11 (2010) 675–679. [5] Y. Sun, H.J. Xu, Y.X. Zhao, L.Z. Wang, L.R. Sun, Z. Wang, X.F. Sun, Crocin exhibits antitumor effects on human Leukemia HL-60 cells in vitro and in vivo, Evid. Based Complement. Altern. Med. 2013 (2013) 690164. [6] S. Ben Jannet, N. Hymery, S. Bourgou, A. Jdey, M. Lachaal, C. Magné, R. Ksouri, Antioxidant and selective anticancer activities of two Euphorbia species in human acute myeloid leukemia, Biomed. Pharmacother. 90 (2017) 375–385. [7] C.S. Lima, D.G. Spindola, A. Bechara, D.M. Garcia, C. Palmeira-Dos-Santos, J. Peixoto-daSilva, A.G. Erustes, L.F.G. Michelin, G.J.S. Pereira, S.S. Smaili, E. Paredes-Gamero, A.K. Calgarotto, C.R. Oliveira, C. Bincoletto, Cafestol, a diterpene molecule found in coffee, induces leukemia cell death, Biomed. Pharmacother. 92 (2017) 1045–1054.
99
Biomedicine & Pharmacotherapy 99 (2018) 96–100
K. Zhang et al.
damage assessed by the cytogenetic tests, Toxicol. Ind. Health 28 (2012) 648–654. [13] S.C. Nair, M.J. Salomi, C.D. Varghese, B. Panikkar, K.R. Panikkar, Effect of saffron on thymocyte proliferation, intracellular glutathione levels and its antitumor activity, Biofactors 4 (1992) 51–54. [14] H. Hosseinzadeh, F. Shamsaie, S. Mehri, Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal, Pharmacogn. Mag. 5 (2009) 419–424. [15] M.R. Salahshoor, M. Khazaei, C. Jalili, M. Keivan, Crocin improves damage induced by nicotine on A number of reproductive parameters in male mice, Int. J. Fertil. Steril. 10 (2016) 71–78.
[8] K. Premkumar, C. Thirunavukkarasu, S.K. Abraham, S.T. Santhiya, A. Ramesh, Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice, Hum. Exp. Toxicol. 25 (2006) 79–84. [9] N. Khan, F. Afaq, H. Mukhtar, Cancer chemoprevention through dietary antioxidants: progress and promise, Antioxid. Redox Signal. 10 (2008) 475–510. [10] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantization of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1998) 184–191. [11] G. Bullock, S. Ray, J.C. Reed, S. Krajewski, A.M. Ibrado, Y. Huang, K. Bhalla, Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL60 cells possessing disparate levels of Bcl-2 protein, Leukemia 10 (1996) 1731–1740. [12] H. Türkez, E. Aydin, The protective role of ascorbic acid on imazalil-induced genetic
100