Silver nanoparticles provoke apoptosis of Dalton’s ascites lymphoma in vivo by mitochondria dependent and independent pathways

Silver nanoparticles provoke apoptosis of Dalton’s ascites lymphoma in vivo by mitochondria dependent and independent pathways

Colloids and Surfaces B: Biointerfaces 136 (2015) 1011–1016 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal...

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Colloids and Surfaces B: Biointerfaces 136 (2015) 1011–1016

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Silver nanoparticles provoke apoptosis of Dalton’s ascites lymphoma in vivo by mitochondria dependent and independent pathways Joe Antony Jacob, Achiraman Shanmugam ∗ Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli-24, Tamil Nadu, India

a r t i c l e

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Article history: Received 3 June 2015 Received in revised form 31 October 2015 Accepted 2 November 2015 Available online 10 November 2015 Keywords: AgNPs DAL Antitumor Apoptosis Molecular mechanism

a b s t r a c t The aim of this report was to investigate the antitumor and apoptotic effects of silver nanoparticles (AgNPs) on the Dalton’s ascites lymphoma cells in vivo. Thirty Swiss albino male mice were assigned into five groups of six each. Group I were intact animals. Group II animals served as tumor control injected with DAL cells intraperitonially. Group III induced animals received plant extract (17 mg/kg BW) and Group IV induced animals received AgNPs (35 ␮g/kg BW). Group V induced animals received standard anticancer drug 5-Fluorouracil (5-FU, 20 ␮g/kg BW). The treatment period was 10 days excluding the day of tumor injection. Tumor cells were collected after euthanizing the animals and real-time PCR was used to analyze p53, caspase-3, 8, 9, 12 and cytochrome C expressions. Results indicate that the AgNPs were efficient in prolongation of life span, reduction of tumor volume and body weight in tumor animals. All the apoptotic genes were upregulated by treatment with AgNPs. To conclude, the present study elicits that AgNPs are potent in antitumor activity and the molecular mechanism is by the induction of apoptosis through the mitochondrial dependent and independent pathways. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Although cancer is a preventable, it is still an obscure goal [1]. Nanotechnology is a multifaceted arena which covers an array of devices designed from various fields of science that are used as nanoscale vectors for targeted delivery of anticancer drugs [2]. To minimize the limitations faced by conventional therapy, research has emerged recently with particular concern over nanomedicine for cancer treatment [3]. Various drug delivery systems have been developed on the nano-regime such as DOXIL (liposomal doxorubicin) and Abraxane (albumin bound paclitaxel) which are already in the market [4]. The conventional chemotherapeutic drugs target cells that undergo rapid division and hence can also cause damage to healthy rapidly dividing cells [5]. Setting aside these limitations, nanotechnology has offered selective targeting of cancer cells. Nanoformulations can actively or passively (through enhanced permeability and retention, called EPR effect) target the tumor cells [6]. Controlled drug delivery is a key issue and novel drug delivery systems have satisfied this concern to a great extent. Taken alto-

∗ Corresponding author. Fax: +91 431 240 7045. E-mail addresses: [email protected], [email protected] (A. Shanmugam). http://dx.doi.org/10.1016/j.colsurfb.2015.11.004 0927-7765/© 2015 Elsevier B.V. All rights reserved.

gether, a nanoformulation must have the capacity to incorporate a drug, preserve it and distribute it to the targeted site [7]. The approval of cisplatin as an antitumor drug has led to the formulation of various other metals as anticancer agents [8]. Among metals and their nanoparticles, AgNPs have received much attention of late due to their biomedical properties such as antiangiogenic, antiproliferative and anticancer effects [9]. Silver is a less toxic metal with regard to biomedical applications in comparison to other metals [10]. Hence, it was used in this study to determine the antitumor activity against DAL cells in vivo. Although these criteria have been taken into consideration and the antitumor effects have been observed by various studies, the molecular mechanism of antitumor activity is still unclear [11]. Hence, this study would be a pioneer in determination of the molecular mechanism of antitumor activity of silver nanoparticles.

2. Material and methods 2.1. Biosynthesis and characterization of AgNPs The biosynthesis and characterization of AgNPs using Rhizophora apiculata has been reported in our previous publication [12]. Further, the concentration of the biosynthesized AgNPs was determined by inductively coupled plasma-optical emission spectroscopic (ICP-OES) analysis undertaken on a JY ULTIMA 2C model.

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2.2. Ex vivo analysis DAL cells were mixed with PBS in the ratio 1:6. The mixture was treated with AgNPs at varying concentrations (25, 50, 75, 100 ␮L) and incubated for varying durations ranging from 3 to 12 h. After incubation, an equal volume of tryphan blue was added and cells were observed using a hemocytometer at 40× magnification for viability.

Table 1 Primers used for analysis of gene expression. Primers

Sequence of the nucleotides

p53

Forward 5 -CACGTACTCTCCTCCCCTCAAT-3 Reverse 5 -AACTGCACAGGGCACGTCTT-3 Forward 5 -TATCCCTGGTCGGTTGAATC-3 Reverse 5 -GGCATAAAACCGTGATTTACA-3 Forward 5 -TCAGTGACGTCTGTGTTCAGGAGA-3 Reverse 5 -TTGTTGATGATGAGGCAGTAGCCG-3 Forward 5 -GCACATTCCTGGTCTTTATGTCCC-3 Reverse 5 -GCCACTGCTGATACAGATGAGGAA-3 Forward 5 -ACA AGGGCATCATCTATGGCTCTGA-3 Reverse 5 -CCAGTGAAGTAAGAGGTCAGCTCAT-3 Forward 5 -TTCATTATTCAGGCCTGCCGAGG-3 Reverse 5 -TTCTGACAGGCCATGTCATCCTCA-3

Cytochrome C Caspase 9 Caspase 12

2.3. Experimental animals Male Swiss Albino mice (weighing 30–35 gm) were purchased and acclimatized to laboratory conditions by housing them in a temperature controlled room a week before commencement of the experiment. The animals were fed with standard feed and water ad libitum during the period. Permission was obtained from Institutional Animal Ethics Committee and CPCSEA regulations were followed during the study. 2.4. DAL cells DAL cells were obtained from Amala Cancer Research Centre, Thrissur, Kerala, India. They were maintained in Swiss Albino Mice in vivo by intraperitoneal inoculation of 106 cells per mice. After the desired volume of cells was obtained, animals were dissected and the experiment was started. 2.5. Experimental design Thirty Swiss albino mice were assigned into five groups of six mice each. Group I were intact animals. Group II animals served as tumor control injected with DAL cells (106 cells per mouse) intraperitonially. Group III tumor induced animals received plant extract (17 mg/kg BW) and Group IV tumor induced animals received AgNPs (35 ␮g/kg BW). Group V tumor induced animals received standard anticancer drug 5-Fluorouracil (5-FU, 20 ␮g/kg BW). The treatment period was 10 days excluding the day of tumor injection.

Caspase 8 Caspase 3

2.9. Real-time analysis of mRNA of apoptotic genes by PCR TRIZOL (Sigma–Aldrich, St. Louis, MO, USA) was used to extract total RNA from tumor cells collected after dissection. Following extraction, RNA concentration and purity were determined. 1 ␮g total RNA from each sample was used to generate cDNA using SuperScript II reverse transcriptase enzyme (Genetech, RT-PCR mix- Germany) in an Agilent amplicon system (AGILENT Biosystems). Real-time PCR was executed by using SYBR Green dye (Invitrogen, Austin, TX, USA) run with a Rotor-Gene 3000 realtime PCR apparatus (Corbett Research, New South Wales, Australia) according to the manufacturer’s instructions. The amount of ␤actin cDNA was used to normalize the quantity of target gene. The value in relation to the control sample was given as 2−CT [13–15]. Real-time PCR primer sequences used in the study are given in Table 1. 2.10. Statistical analysis All results were expressed as mean ± SD. One-way ANOVA to indicate significant variance was performed by using SPSS version 17 (SPSS Inc., Chicago, IL, USA) and individual comparisons were obtained by LSD [16]. Statistical significance was set at p < 0.05. 3. Results and discussion

2.6. Analyses of blood Blood was collected from animals after the experimental period by cardiac puncture, anticoagulated by heparin and used for hematological assay. Fibrinogen free serum was obtained by centrifuging the collected blood at 1000 rpm for 20 min. The obtained serum was used for estimation of serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), calcium and lactate dehydrogenase (LDH). 2.7. Analyses of liver For antioxidant assays, the liver was excised, homogenized with 0.1 M phosphate buffer (pH 7.4) and the homogenate was centrifuged at 3000 rpm for 15 min. The supernatant was collected and centrifuged at 12,000 rpm for 30 min and used to determine the levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA). 2.8. Histopathology Liver tissue was collected and fixed in formalin (10%) and paraffin sections were prepared with thickness of 5 ␮m and stained with hematoxylin and eosin. The pathological changes were observed under a microscope.

AgNPs are reported to possess antitumor activity against lymphoma and melanoma cell lines in vivo [17,18]. Based on this, we used R. apiculata derived AgNPs for antitumor activity against DAL in vivo. Trace metal identification by ICP-OES at the characteristic wavelength for silver, 328.068 nm, showed that the concentration of the metal was 19.51 ␮g/mL. Ex vivo analysis showed that 50 ␮L was the IC50 value of plant extract and AgNPs (as per ICP-OES) which was 17 mg/kg and 35 ␮g/kg BW of mice respectively. The particle size of the AgNPs synthesized using R. apiculata as reported in our earlier publication ranges between 19 and 42 nm [12]. DAL, a tumor of peritoneal cavity includes the ascitic fluid which is the nutritional fluid of the tumor-bearing animals [19]. The body weight and tumor volume of the animals treated with AgNPs decreased considerably in comparison to the control group. The life span increased from approximately 14 days in control group to 21 days in case of AgNPs treatment. This is an increase of 46.52% in life span or survival, which is quite significant in considering the efficacy of an antitumor drug (Fig. 1). An increase in life span and reduction in tumor volume are reliable criteria for application of an anticancer drug [20,21]. Since the tumor growth parameters returned to normal levels, AgNPs were found to be effective in vivo against lymphomagenesis. Anemia, a reduction in RBC or hemoglobin levels is a prognostic factor in the case of lymphoma [22]. Cancer-associated anemia is a common phenomenon that occurs with cancer progression [23].

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Fig. 1. Antitumor parameters after treatment with biosynthesized AgNPs. Results are expressed as mean ± SD (n = 6). Each lower case letter above the error bars indicate the significant (p < 0.05) difference between the groups.

Table 2 Biochemical parameters of study. Results are expressed as mean ± SD (n = 6). Each lower case letter above the standard deviation indicate the significant (p < 0.05) difference between the groups. Parameters

Groups I

II

III

IV

V

Cellular metabolite LDH (IUdL−1 )

1204 ± 126.53c

3739 ± 441.75a

1834 ± 144b

1500 ± 126.25c

1459 ± 154.49c

Lipid peroxidation marker MDA (nmoles/gm)

1.042 ± 0.14d

5.237 ± 0.34a

3.232 ± 0.66b

1.936 ± 0.27c

2.112 ± 0.62c

Enzymatic antioxidant SOD (IU/mg)

8.65 ± 1.01a

5.566 ± 0.82c

6.937 ± 0.79b

7.895 ± 0.85ab

7.725 ± 0.68ab

Non-enzymatic antioxidant GSH (IU/mg)

5.967 ± 0.49c

3.401 ± 1.04c

4.273 ± 0.61bc

5.124 ± 0.49ab

4.976 ± 0.52b

Liver enzymes SGOT (IU/l) SGPT (IU/l) ALP (IU/l)

254 ± 10.2c 48.4 ± 1.46d 81.05 ± 2.29d

553 ± 32.22a 65.84 ± 3.60a 142.6 ± 2.15a

325 ± 31.9b 61.3 ± 3.83b 97.3 ± 2.97b

300 ± 21.2b 44.5 ± 2.79e 80.1 ± 2.87d

317 ± 14.8b 54.3 ± 1.99c 87.4 ± 2.91c

Hematology RBC (cells/cu.mm) WBC (cells/cu.mm) Hb (gm%)

3.3 ± 0.60a 3800 ± 231.55d 9.8 ± 0.81a

1.6 ± 0.25c 5800 ± 230.5a 4.5 ± 0.49c

2.4 ± 0.49b 4650 ± 248.2b 8.2 ± 0.40b

2.7 ± 0.40ab 4100 ± 388.5cd 9.2 ± 0.50a

2.8 ± 0.31ab 4250 ± 203.1c 9.1 ± 0.55a

Leukocytosis, or elevated WBC count is a determinant of inflammation and an important characteristic associated with cancer [24]. As a prognostic tool, the results of this study also coincide with the observations of previous studies as mentioned above. Speculations made on the mechanism of antitumor activity of various agents are by two means: one by directly acting on the tumor and the other by means of acting on angiogenesis [25]. Vessel

growth was observed in case of control animals, whereas vessel regression or control in angiogenesis was observed in case of AgNPs treated group (data not shown). Tumoral angiogenesis, the process of formation of new blood vessels from pre-existing ones, is a key factor in growth and spread of tumor cells. Oxidative stress plays a key role in angiogenesis and hence the antioxidant property may

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Fig. 2. Histoarchitecture of liver of R. apiculata derived AgNPs treated groups.

Fig. 3. Assay for LDH and Hypercalcemia. Results are expressed as mean ± SD (n = 6). Each lower case letter above the error bars indicate the significant (p < 0.05) difference between the groups.

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Fig. 4. Molecular mechanism of antitumor activity of AgNPs.

be vital for antiangiogenic activity [10,26]. Hence we further tested the antioxidant enzymes. Disorder in antioxidant systems can occur due to tumor growth [27]. SOD is a key enzyme in defense against oxidative stress and is a prospective target for cancer therapy [28]. The conversion of superoxide to H2O2 is catalyzed by SOD [29]. Tumor growth is known to suppress the levels of SOD [30]. Similar findings were observed in our study, where SOD levels were decreased in the tumor induced group and brought back to near normal levels in treatment groups. GSH plays foremost role in cellular processes such as cell differentiation, proliferation, and apoptosis. Decreased levels of GSH lead to impairment of cellular homeostasis and are associated with diseases such as cancer [31]. These antioxidants efficiently inhibit tumor growth and this might be credited to the combinatorial effect of antioxidant and antitumor activity of the AgNPs. Impairment of redox homeostasis has been reported to be a key factor in a variety of neoplasms. MDA, a major reason for lipid damage is a primary index for lipid peroxidation in vivo which is over-expressed in case of cancers [32,33]. Due to development of tumor, peroxidative damage on the liver has occurred, which has resulted in increased levels of MDA, which reverted to normal with treatment. Since elevated levels of MDA were observed, the possible negative effects on liver are evident. Therefore we analyzed the liver function tests. Hematological malignancies such as lymphoma, cause malignant cells to permeate the liver and result in abnormalities in liver function tests [34]. Elevation of aminotransferases and ALP may occur due to infestation of liver by tumor [35]. The elevated levels of serum enzymes therefore indicate the injury to hepatocytes [36]. The leakage of liver enzymes will result in increased concentrations in serum, as a marker of hepatic injury [37]. Leakage of serum enzymes was evident in tumor induced group as liver

enzymes were elevated. This was brought back to near normal levels in treatment groups (Table 2). Since the liver enzymes were elevated in the control group, possible injury to liver is apparent. Hence we further analyzed histomorphological changes. Histology of liver sections of normal animals exhibited hepatic cells with a well-defined cytoplasm, prominent nucleus and a well defined central vein. Control group animals showed total loss of hepatic architecture with centrilobular hepatic necrosis, vacuolization and disintegrated central vein. These effects were revitalized to normal architecture in treatment groups (Fig. 2). The transformation of mild forms of lymphoma to aggressive forms is usually characterized by elevated levels of LDH and hypercalcemia [38]. This was evident in control groups, where the LDH levels were increased and hypercalcemia was observed (Fig. 3). Hypercalcemia observed in tumor-induced groups is probably due to the reason that it is common in 10–30% of cancer patients at the later stage of the disease [39]. Cancer is ranked top-most among the reasons for hypercalcemia. During lymphoma, calcitriol is produced in large quantities, which results in increased calcium absorption and reabsorption in the intestines and kidneys leading to hypercalcemia [40]. In the case of T-cell lymphoma such as DLA, serum LDH levels tend to be elevated which could be considered as a prognostic marker [41,42]. Cancer cells are more acidic compared to normal cells as they release lactic acid. LDH catalyzes the conversion of pyruvate (a product of glycolysis) to lactic acid. So there will be higher levels of lactate in case of metastatic cancer when LDH is higher and therefore the cancer cells will be more acidic [43,44]. This correlates with the results obtained in this study. When tumor cells become resistant to antitumor therapy, the major reason would be the defects caused in initiation of apoptosis [45]. This is due to the fact that antitumor agents prevent the development of tumor by apoptotic induction [46]. Apoptosis

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is a resolutely synchronized process, with major effectors being the family of caspase cascade [47]. Apoptosis is usually triggered by external cues and factors from mitochondria which result in the extrinsic death receptor and intrinsic mitochondrial pathways respectively [48]. The extrinsic pathway is usually instigated by the ligation of death receptors such as Fas, TNF and TRAIL to the activator caspase-8 which activates the protease by induced proximity model. This leads to an increase in cytotoxic stimuli, resulting in activation of the effector caspase-3 [49]. Intrinsic pathway through activation of p53, results in the release of cytochrome C from mitochondria by causing disruption of its membrane leading to activation of caspase-9 [50,51]. In the intrinsic pathway, the ER stress can also induce apoptosis by activation of caspase-12 as an initiator caspase [52]. The extrinsic and intrinsic apoptotic pathways ultimately lead to activation of effector caspase-3 [53]. The activation of downstream caspase-3 leads to cell death [54]. The possible mechanism behind the antitumor activity of AgNPs has been attributed to the involvement of caspases [55]. Here in this study, by AgNPs treatment, caspase-3, 8, 9, 12, p53 and cytochrome C were all upregulated (Fig. 4, Supplementary material 1 and 2). This is due to the fact that AgNPs might have led to the activation of p53, which in turn led to the intracellular release of cytochrome C. This might have led to the activation of caspase-9. The formation of DISC complex by death receptors also results in activation of caspase-8. The activation of upstream caspases-9 and 8 has led to the activation of downstream caspase-3, which ultimately led to cell death. This demonstrates that the mechanism of antitumor activity of AgNPs was by the involvement of p53 dependent intrinsic and extrinsic pathways. 4. Conclusion To the knowledge of authors, this is the first ever report made on determination of the molecular mechanism of antitumor activity of biosynthesized AgNPs in vivo which is by the induction of apoptosis by both the mitochondrial dependent and independent pathways. Tumor growth, hematological, biochemical parameters were brought back significantly to near normal levels by treatment with AgNPs. The results indicate that the antioxidant activity of AgNPs could be a possible reason behind the antitumor activity of AgNPs. Acknowledgements The authors acknowledge the support for research by the grants of University Grant Commission (UGC), Council for Scientific and Industrial Research (CSIR) and Department of Science and Technology (DST), New Delhi, Government of India. The authors also gratefully acknowledge Dr. Rachel Adams, Senior lecturer, Cardiff School of Health Sciences, Cardiff Metropolitan University, for language correction of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.11. 004. References [1] V.T. De Vita, S.A. Rosenberg, N. Engl. J. Med. 366 (2012) 2207. [2] M. Ferrari, Nat. Rev. Cancer 5 (2005) 161. [3] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nat. Nanotechnol. 2 (2007) 751.

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