Quercetin alleviates myocyte toxic and sensitizes anti-leukemic effect of adriamycin

Abstract

Objectives

Derived from plants, flavonoids have been proven to possess anti-cancer activities. Adriamycin (ADM), an anthracycline antibiotic, is widely applied in the chemotherapy for leukemia; however, it has a side effect of heart damage. This study aims to explore potential anti-leukemia effects of quercetin (Que) and the underlying mechanism.

Methods

The P388 xenograft mice models were first established and then treated with Que alone or in combination with ADM. Subsequently, we evaluated their effects on cell proliferation and apoptosis by observing the cell cycle and detecting the Caspase-3 level, respectively. The underlying pro-apoptotic mechanism was further investigated by detecting the expression levels of NF-κB, Bcl-2, and Bax. The cardiomyocyte ultrastructural changes of P388 leukemic mice after drug treatment were also observed. The protective effect of Que on cardiomyocyte was evaluated by detecting enzymatic activity changes of glutathione peroxidase, superoxide dismutase, and malondialdehyde.

Results

Compared with ADM group, the combination of ADM and Que showed prolonged survival time and less peripheral white blood cells. Que could sensitize the anti-leukemic effect of ADM by inhibiting the proliferation of white blood cells through trapping the cells at the S phase; caspase-3 was activated via the expressional regulation of Bcl-2, Bax, and NF-κB. When applied in combination with ADM, Que could attenuate heart damage by cleaning the reactive oxygen species.

Conclusion

Our study may provide informative evidences for the underlying mechanism of anti-cancer effects of Que and sheds light on the clinical application of Que in leukemia treatment.

Keywords:

• Leukemia
• Xenograft nude mice
• Quercetin
• Adriamycin
• Myocyte damage

Introduction

Leukemia is a blood or bone marrow cancer type with abnormal increase of immature white blood cells.¹ Clinically and pathologically, leukemia is subdivided into acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, and chronic myelogenous leukemia. Leukemia patients often display impaired blood clotting process, weak immune system, and neurological symptoms.² Every year ∼256 000 people around the world develop some form of leukemia, and 209 000 people die from it.³ About 90% of the leukemias are diagnosed in adults. Most forms of leukemia can be treated by chemotherapy, medical radiation therapy, hormone treatments, or bone marrow transplant.^4,5 Adriamycin (ADM), an anthracycline antibiotic, is widely applied in chemotherapy of many cancer types, including leukemia.⁶ ADM works by intercalating DNA.^7,8 However, ADM had the most dangerous side effect of heart damage,^9–11 which is due to the myocyte damage caused by reactive oxygen species (ROS) generated from the interaction between ADM and iron.

Fruits and vegetables contain many bioactive compounds, which have been proven to be responsible for reducing the risk of common human cancers. Increasing interest has been paid to flavonoids, a large group of compounds with a similar structure, which consists of two phenolic benzene rings linked to a heterocyclic pyre or pyrone.¹² Recently, quercetin (Que), (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one), a kind of flavonoid, has been reported to possess anti-cancer effects.¹³ It has been reported that the uptake of Que has an inverse association with the incidence of pancreatic cancer and lung cancer in current smokers.¹⁴ Although there is no direct evidence for the interaction of Que with cellular receptors, various cellular receptors have been proven to be involved in the cancer-preventive property of Que, such as the 67 kDa cell surface laminin receptor,¹⁵ insulin-like growth factor-I,¹⁶ vimentin,¹⁷ and an aryl hydrocarbon receptor.^18,19 Que also modifies signal transduction during carcinogenesis, including cell cycle regulation, apoptosis, pro-inflammatory protein induction, and angiogenesis.

However, there is no relative study about potential effects of Que on leukemia treatment. Here, we investigated the anti-leukemia effects of Que and its potential sensitization effects to ADM in a P388 xenograft nude mice model. We also investigated the underlying mechanism by determining the levels of Caspase-3, Bcl-2, Bax, NF-κB, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA). Our study may provide informative evidences for the mechanism exploration of the anti-cancer effects of Que and sheds light on the clinical application of Que in leukemia treatment.

Materials and methods

All animal studies have been approved by The Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University and performed in accordance with the ethical standards.

Cell culture

P388 leukemia cells (Ruiqi Biotechnology Company, Shanghai, China) were cultured in RPMI1640 (Hyclone, Shanghai Bioleaf Biotech Co., Ltd, Shanghai, China) medium containing 10% fetal bovine serum (Hyclone) at 37°C in 5% CO₂ (incubator: Thermo Electron Corporation, Cambridge, UK; ultra-clean bench: Beijing Guanpeng Purification Equipment Co., Ltd, Beijing, China). The cells were collected at the exponential phase.

Leukemia mice model design

Seventy SPF Balb/c nude mice (35 males and 35 females) were purchased from Beijing Vital River Laboratories Company (certificate No.: SCXK 2006-0009) and raised in SPF biological laminar flow rack at the Clinical Medical Research Center of the Affiliated Hospital of Inner Mongolia Medical University.

The P388 cells at the exponential phase were collected and washed with phosphate-buffered saline (PBS; pH 7.4) twice, then re-suspended (refrigerated centrifuge: Sigma, Osterode, Germany) with PBS at 10⁶ cell/ml, and finally subcutaneously injected into the left anterior axilla of the mice (0.2 ml for each).

Experimental grouping and medication method

Que (Sigma, St. Louis., MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Beijing Chemical Reagent Company, Beijing, China). ADM (Shenzhen Main Luck Pharmaceuticals, Inc., Shenzhen, China) was prepared in physiological saline (PS). After the elimination of the mice with no xenograft or abnormal tumor size, the remaining 60 mice with tumor diameter of 0.8 cm were randomly divided into six groups: control, low Que, high Que, ADM, ADM with low Que, and ADM with high Que for peritoneal injection lasting for 15 days (Table 1).

Table 1. Medication methods for six P388 leukemic nude mice groups

Tail blood was collected before and after P388 inoculation at Weeks 0, 2, 3, and 4 from 2 days after medication for peripheral blood hemogram and white blood cell number determination.

Life prolongation rate

The survival time was counted from the day when mice were inoculated. Life prolongation rate and sensitization were calculated according to the following formulae: life prolongation rate = (average survival time of experimental group/average survival time of control group − 1) × 100%; sensitization = average survival time of experimental group/average survival time of control group.

Cell cycle detection assay

Tumor tissues of the mice were minced and centrifuged at the end of Week 4, then, the cell suspension was transferred into a BD flow cytometry tube. The cells were washed with 500 µl 1× PBS and digested with 500 µl 0.25% trypsin. The liquid in the tube was then collected and centrifuged at 2000 rpm, 4°C for 8 minutes. Next, the cells were re-suspended in 1 ml 1× PBS. While gently vortexing the suspension, 100 µl 1× PBS and 900 µl 90% ethanol were added in a drop-wise manner. After fixing at 4°C for 2 hours, the cells were collected after centrifugation and washed with 1 ml 1× PBS. Finally, 300 µl PI dye was used for staining at room temperature for 10–15 minutes. Cell cycle analysis was performed with Flow Cytometry (Beckman Coulter, Inc., Fullerton, CA, USA).

Apoptosis analysis

Tissues were minced, weighted, and homogenized manually in proper volume of, pH 7.4, PBS at the end of Week 4. After centrifugation at 2000–3000 rpm for 20 minutes, the supernatant was carefully collected and used for apoptosis analysis according to the manual of Annexin V-FITC ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The sample concentration was determined by measuring the optical density (OD) value at 450 nm with a microplate reader (Thermo Electron Corporation, Cambridge, UK).

Real-time polymerase chain reaction

The total RNA was extracted according to the manual of Trizol (Tiangen Biotech, Co., Ltd, Beijing, China). The RNA concentration and purity was determined with a trace nucleic acid analyzer. The total RNA (2 µg) was used for reverse transcription (Tiangen Biotech, Co., Ltd) in accordance to the following condition: 20 µl system, 70°C for 5minutes, 2 minutes on ice, 25°C for 10 minutes, 42°C for 50 minutes, 95°C for 5 minutes. The cDNA template was used for real-time PCR (Real-Time PCR kit: Tiangen Biotech, Co., Ltd, ABI7300 Real-Time PCR Instrument: ABI) using the condition below: 10 µl system, 95°C for 10 seconds, 55°C for 20 seconds, 72°C for 31 seconds, 40 cycles. Primers used are listed in Table 2. Using GAPDH as an internal reference, relative contents for Bcl-2, Bax, and NF-κB were calculated using the 2^−△△Ct method: △Ct = Ct_{test gene} = Ct_GAPDH; △△Ct = △Ct (Experimental group) = △Ct (DMSO control group); fold change N = 2^−△△Ct.

Table 2. Real-time PCR primers for Bax, Bcl-2, NF-κB, and GAPDH

Western blotting

The cells were washed twice with cold PBS and applied with 50 µl lysis buffer on ice for 20 minutes. The cells were then collected, and transferred into a pro-cold 1.5 ml EP tube on ice for 1 hour. Subsequently, the total protein supernatant was obtained by centrifugation (10 000 rpm at 4°C for 10 minutes) and used for concentration determination via the Bradford method. Loading samples were prepared by adding an equal volume of 2× sodium dodecyl sulfate buffer and incubating in boiling water for 5 minutes, and then sodium dodecyl sulfate–polyactylamide gel electrophoresis separation was performed at 120 V for 15 hours. Protein bands were transferred onto nitrocellulose membrane (NCM) with electroporator at 58 V, 4°C for 4 hours. The NCM was subsequently incubated at room temperature in 1:200 primary antibodies and 1:500 secondary antibodies for 2 and 1 hour, respectively. All the antibodies (anti-Bcl-2, Bax, NF-κB p65, and β-Ainten) were purchased from Santa Cruz. NCM was developed by adding diaminobenzidine (DAB) substrate for 20–30 minutes. The reaction was terminated by washing the NCM with water. Immunoblotting for the internal reference β-tubulin was conducted simultaneously. The digital images were taken after DAB visualization. The images were analyzed with the GDS8000 Image Analysis System. The average OD value of a certain protein band was used to indicate the protein expression level.

Ultrastructure changes of cardiomyocyte

Two weeks after medication, the myocardial tissue from the left ventricle was minced into <1 mm explants. The samples were fixed in 3% glutaraldehyde and 1% osmic acid, sequentially. After dehydration, fixed explants were embedded with Epon812 and sliced, followed by uranium lead double staining, the myocyte ultrastructural changes were observed under electron microscopy.

GSH-Px activity detection

The 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) colorimetric technique was utilized. One gram fresh myocardium was homogenized in 25 ml of 5 mM EDTA-TCA solution, and then 2 ml of solution was extracted and mixed with 0.4 ml of 1 mM NaOH solution, 1.5 ml of 0.2 M pH 7.0 potassium phosphate buffers, and 0.1 ml of DTNB reagent. The mixture was developed at room temperature for 5 minutes. Distilled water was added to the mixture to 5 ml in total and OD at 412 mm was measured. The blank control was set by replacing DTNB with potassium phosphate buffer. Reduced form of GSH was used as a reference standard.

SOD and MDA activity detection

Mice hearts were first minced and then homogenized with nine volumes of PS. After centrifugation at 3000 rpm for 5 minutes, the supernatant was used to detect SOD content and MDA content with the hydroxylamine method and the thiobarbituric acid method in accordance with the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively.

Data analysis

SPSS13.0 was used for statistical analysis. Data of all the groups were expressed as average value ± standard deviation. Paired comparisons among groups were performed with one-way analysis of variance. P < 0.05 was considered to indicate statistical significance.

Results

Que prolongs the survival time of P388 xenograft Balb/c nude mice

After being inoculated with P388 cells for 2 weeks, obvious bulges were seen underneath the skin of the left anterior axilla (Fig. 1A), and 4 weeks later, the mice displayed different degrees of mental fatigue, accidie, wrinkled fur, plump abdomen, and appearances of xenograft tumor (Fig. 1B). Compared with DMSO control group, low Que, high Que, ADM, ADM with low Que, and ADM with high-Que group showed lifetime prolongation rates and sensitizations of 58.96% and 0.62, 79.10% and 0.56, 57.46% and 0.65, 83.58% and 0.55, and 105.22% and 0.51, respectively (Table 3).

Figure 1. P388 xenograft mice models. (A) Nude mice images taken 2 weeks after P388 cell inoculation; (B) nude mice images taken 4 weeks (B) after P388 cell inoculation.

Table 3. Survival time summary for control and experimental groups

Que could reduce white blood cells of peripheral blood in P388 xenograft Balb/c nude mice

The number of white blood cells in the peripheral blood of P388 leukemic mice had been increasing significantly 2 weeks later since P388 inoculation. After Que and ADM medication, the white blood cell number of each medication group decreased, of which the ADM with high-Que group showed the most significant change (Fig. 2).

Figure 2. White blood cell number of the peripheral blood from each group of P388 leukemic mice. *Significant difference compared with DMSO control group (P < 0.05).

Que changes cell cycle of P388 xenograft Balb/c nude mice

The Flow Cytometry data showed the portion of cells at the G₀–G₁ phase at each of the medication group decreased significantly (P < 0.05), while cells portion at the S phase and the G₂–M phase remarkably increased (P < 0.05) when compared with DMSO control group (Fig. 3). ADM with low-/high-Que groups displayed the most significant changes.

Figure 3. Portions of cells at different cell cycle phases for each group of P388 Xenograft Balb/c nude mice. Left: Cell percentage at G₀–G₁; right: cell percentage at S–G_2M.*Significant difference compared with DMSO control group (P < 0.05).

Que affects apoptosis by regulating Caspase-3 activity

Compared with DMSO control group, low-Que group showed enhanced Caspase-3 level, while high-Que group displayed reduced Caspase-3 level (Fig. 4A). High-Que group had similar low Caspase-3 level as ADM group. There is no significant Caspase-3 level difference between high-Que and low-Que groups when they were applied in combination with ADM.

Figure 4. Que activates Caspase-3 by regulating the expression of BAX, Bacl-2, and NF-κB. (A) Caspase-3 activity comparisons among control group and medication groups. Data were generated from Caspase-3 ELISA; (B) BAX mRNA level in different groups; (C) Bcl-2 mRNA level in different groups; (D) NF-κB mRNA level mRNA level in different groups; (E) BAX, Bacl-2, and NF-κB protein level comparisons among control and medication groups. ^aComparison with DMSO control group P < 0.05; ^bcomparison with low-Que group P < 0.05; ^ccomparison with ADM group, P < 0.05; ^dcomparison to ADM with low-Que group, P < 0.05.

Que could affect apoptosis by regulating the expression of Bcl-2, Bax, and NF-κB p65

Real-time polymerase chain reaction (PCR) results showed that compared with DMSO control group, all the medication group showed enhanced Bax mRNA level (Fig. 4B) and decreased Bcl-2 (Fig. 4C) and NF-κB p65 mRNA (Fig. 4D) levels. The western blot results (Fig. 4E) showed that Bax protein levels of low Que and ADM with high-Que group were slightly increased compared with DMSO control group (P < 0.05), and protein levels of Bcl-2 and NF-κB p65 from the medication groups were reduced significantly in a dose-dependent manner, of which ADM with Que group showed the most significant changes (P < 0.05).

Que could alleviate the side effect of ADM by protecting cardiomyocyte

Transmission electron microscopy (TEM) images showed that the ADM group had series of cardiomyocyte damages, such as uneven myocardial fibers arrangement, fuzzy boundaries, myocardial fiber disorders, break-outs, disintegrations, and increased mitochondrial vacuolars (Fig. 5B) compared with DMSO control group (Fig. 5A). When ADM was applied with Que, the damages to cardiomyocytes were alleviated to some degree (Fig. 5C).

Figure 5. Ultrastructural changes of cardiomyocyte after ADM medication. Images were taken under TEM (×120 00). (A) DMSO control group; (B) ADM group; and (C) ADM with low-Que group.

Que regulates activities of GSH-Px, SOD, and MDA

There was no difference between DMSO control and ADM group in terms of GSH-Px (Fig. 6A), SOD (Fig. 6B), and MDA (Fig. 6C) activities. Except ADM group, all the Que groups had higher GSH-Px and SOD activities, and lower MDA activity when compared with either DMSO control or ADM group.

Figure 6. Que removes ROS by regulating GSH-Px, SOD, and MDA activities. (A) GSH-Px enzymatic activity comparisons among all groups; (B) SOX enzymatic activity comparisons among all groups; (C) MDA enzymatic activity comparisons among all groups. ^aComparison with DMSO control group, P < 0.05; ^bcomparison with ADM group, P < 0.05.

Discussion

With generally less hazards to safety than synthetic compounds, there is a growing trend and increasing market for natural bioactive compounds extracted from fruits and vegetables. Botanical products are being used as functional foods, food supplements, flavors, and cosmetics. There are laboratory studies investigating Que's potential for anti-cancer applications.^{12,13,20–24} But so far there have been still limited in vivo studies on the anti-cancer effects. By using the model of P388 xenograft leukemic nude mice, we provided strong in vivo evidences that Que could promote cell apoptosis by regulating cell cycle. Bcl-2 is the founding member of the Bcl-2 family of regulator that inhibits apoptosis.^25,26 NF-κB, a protein complex that controls transcription of DNA, is found in almost all animal cell types and involved in immune response,²⁷ cell proliferation, and cell survival.²⁸ Increased susceptibility to apoptosis caused by NF-κB defection could increase cell death. NF-κB regulates anti-apoptotic genes of TRAF1 and TRAF2; therefore, it can inhibit the activities of the caspase enzymes, which are central to most apoptotic processes.²⁸ Our results showed that Que could promote apoptosis by inhibiting the expression of NF-κBp65 and Bcl-2, and increasing Caspase-3 activity and cell apoptosis, which are consistent with the mechanistic framework generated so far.

Since its discovery from Streptomyces peucetius in the 1950s and clinical trials in the 1960s, ADM has been used as an important chemotherapeutic drug for a broad range of cancer types, such as leukemias, Hodgkin's lymphoma, bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, and multiple myeloma. However, when the cumulative dose reaches 550 mg/m², there will be a high risk of developing cardiac side effects.²⁹ The cardiotoxicity of ADM is caused by a dose-dependent decline in mitochondrial oxidative phosphorylation.³⁰ What is more, the ROS generated by the interaction between ADM and iron will cause myofibrillar loss and cytoplasmic vacuolization. Our results indicated that Que could effectively alleviate the cardiomyocyte damages caused by ADM. We further found out Que treatment could increase the GSH-Px and SOD activities, which are the most important enzymes to clean the intracellular ROS and protect the organism from oxidative damage. Some reports pointed out that efficient antioxidant activity of Que is related to its reducing properties, such as a 3′,4′-dihydroxy(=catechol) moiety, a C4 = O keto-group, a 3-hydroxyl substituent, and a C2 = C3 double bond.³¹ Interestingly, Que could also promote the expression of the enzymes to clean the ROS. More investigations are needed to reveal how Que triggers the signaling pathways.

In spite of the anti-cancer aspects, Que may be toxic, genotoxic, and carcinogenic. The mutagenic properties of Que have been demonstrated in a variety of bacterial and mammalian mutagenicity tests, and have been related to its quinone/quinone methide chemistry.^32,33 There is a long way to go in terms of clinical use of Que, and future efforts are needed to estimate the biological safety of Que. Structure modification of Que might be useful to alleviate or eliminate the genotoxicity of Que.

In conclusion, by using the P388 xenograft nude mice model, our study showed that Que significantly prolonged the survival time of P388 leukemic mice by inhibiting the proliferation of white blood cells by trapping them at the S phase. Que also promoted cell apoptosis by activating Caspase-3, which is caused by suppressed expression of Bcl-2 and NF-κB. Que could also attenuate the heart damage caused by ADM through cleaning the ROS. Our study may provide strong evidences for the underlying mechanism of anti-leukemic effect of Que and sheds light on the clinical application of Que in leukemia treatment.

Acknowledgment

We wish to express our warm thanks to Fenghe (Shanghai) Information Technology Co., Ltd. Their ideas and help gave a valuable added dimension to our research.

Disclaimer statements

Contributors YH and HY participated in the design of this study, and they both performed the statistical analysis. JW carried out the study, together with YR, collected important background information, and drafted the manuscript. XS and YS conceived of this study, and participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.

Funding The National Natural Science Foundation of China (81041045).

Conflicts of interest None.

Ethics approval All animal studies have been approved by The Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University and performed in accordance with the ethical standards.