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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 3  |  Issue : 1  |  Page : 5-10

Plasma from breast cancer patients inflicts injury to cord-derived stem cells in vitro


Department of IMBB, The University of Lahore, Lahore, Pakistan

Date of Web Publication29-Feb-2016

Correspondence Address:
Dr. Nadia Wajid
The University of Lahore, Defense Road Campus, Lahore
Pakistan
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Source of Support: None, Conflict of Interest: None


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  Abstract 

Aims and Background: Stem cells derived from Wharton's jelly-derived mesenchymal stem cells (WJMSCs) hold a great therapeutic potential for tissue repair and regeneration and act as a vehicle for gene therapy of multiple human diseases including cancer. The study aims to find the growth of WJMSCs in breast cancer plasma to evaluate their potential in breast cancer therapy. Materials and Methods: WJMSCs at passage 3 were cultured in the plasma isolated from breast cancer patients keeping healthy persons as controls. The effects on cell viability, proliferation, cell death rate, and paracrine factors (VEGF, p53, and p38) were assessed by crystal violet staining, MTT assay, lactate dehydrogenase (LDH) assay, and enzyme-linked immunosorbent assay (ELISA), respectively. Oxidative stress was assessed by the estimation of free radical species malondialdehyde (MDA) and enzymes glutathione (GSH), catalase, and superoxide dismutase (SOD). Results: It was observed that WJMSCs cultured in the plasma of breast cancer patients exhibit decreased viability, slow proliferation, high LDH release, low expression of VEGF while there are high expressions of p38 and p53, and a high oxidative stress. Conclusion: In conclusion, our study demonstrates that a stressed environment of cancer plasma induces injury to the WJMSCs and reduces their viability. The efficacy of these cells for the therapeutics of cancer is reduced in cancer conditions, which necessitates further studies to improve cell survival in cancer conditions.

Keywords: Breast cancer, cell death, oxidative stress, proliferation, Wharton′s jelly mesenchymal stem cells (WJMSCs)


How to cite this article:
Wajid N, Javed S, Ali F, Ali M, Anwar SS. Plasma from breast cancer patients inflicts injury to cord-derived stem cells in vitro. Sifa Med J 2016;3:5-10

How to cite this URL:
Wajid N, Javed S, Ali F, Ali M, Anwar SS. Plasma from breast cancer patients inflicts injury to cord-derived stem cells in vitro. Sifa Med J [serial online] 2016 [cited 2024 Mar 29];3:5-10. Available from: https://www.imjsu.org/text.asp?2016/3/1/5/177685


  Introduction Top


Breast cancer is one of the prevalent cancers with more than 1,300,000 incidences around the globe. [1] It is the most common female malignancy and the second leading cause of death by cancer with 23% mortality rate among all cancers. [2] Early diagnosis as well as increased use of adjuvant and neoadjuvant therapies has increased the survival of breast cancer patients over the previous decades but the death rate is still very high due to recurrence leading to metastasis. Treatment options available for breast cancer include targeted anti-human epidermal growth receptor 2 (HER2) therapy, chemotherapy, antiangiogenic therapy, and hormonal therapy. [3]

Autologous stem cells transfusion is also employed after chemotherapy of breast cancer patients for replacement of damaged bone marrow as chemotherapy damages vital hematopoietic stem cells. [4] Mesenchymal stem cells (MSCs) have the ability to migrate to tumors, leading to incorporation in the tumor stroma. These properties make them promising vehicles for gene therapy in multiple metastatic cancers including breast cancer. [5] MSCs from the adipose tissue and bone marrow have been shown to directly produce proapoptotic agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) after gene therapy. [6],[7]

Wharton's jelly mesenchymal stem cells (WJMSCs) have been demonstrated to be beneficial in several preclinical animal models for various diseases such as neurodegenerative diseases, heart diseases, and cancer via trophic rescue and immune modulation. [8] These cells have been reported to inhibit cancer both in vivo and in vitro by multiple mechanisms leading to various phenotypic changes such as cell shrinkage, fragmentation, blebbing, vacuolation, membrane damage, and acquisition of granular particles. [9]

The present study was designed to evaluate the effects of breast cancer patients' plasma on the viability and growth of WJMSCs to estimate the potential effects if these cells are to be transplanted in patients. It has been hypothesized that stress in the blood of cancer patients may affect the therapeutic potential of WJMSCs for breast cancer.


  Materials and Methods Top


Collection of breast cancer plasma

Plasma samples from 50 females who were breast cancer patients and 50 healthy females were collected from INMOL Hospital, Lahore, Punjab, Pakistan with the consent. This study was approved by the local Ethics Committee at The University of Lahore in Lahore, Punjab, Pakistan.

Procurement of human umbilical cord

The present study was approved by the Biosafety Board at the University of Lahore, Lahore, Punjab, Pakistan. The others selected for the study were negative for human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). Five umbilical cords were obtained with the consent of the parents from full-term caesarian sections.

Isolation, culturing, and characterization of WJMSCs

WJMSCs from the umbilical cord were isolated, as previously reported. [10] Briefly, the cord pieces were incubated in 3 mg/mL collagenase solution (Invitrogen Inc., Carlsbad, CA, USA). After 3 h, Dulbecco's modified eagle medium-low glucose (DMEM LG) (Sigma Aldrich, USA) with 10% fetal bovine serum (FBS) (Sigma Aldrich, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, Missouri, USA) was added to the same T75-flasks (Corning Inc., Corning, NY, USA) containing collagenase solution. The medium was renewed after 3 days. Cells were used at passage 3 for all further experiments. The cells were characterized by fluorescence-activated cell sorting (FACS) analysis (see Wajid et al.). [10]

Treatment of WJMSCs with breast cancer plasma

WJMSCS were plated at a density of 100,000 cells per well of a 6-well plate (Corning, USA) and cultured for 24 h and subsequently treated with DMEM LG, along with 10% human plasma from normal persons (Group 1) or from breast cancer patients (Group 2). After 3 days of culturing, the medium was saved at 80°C for lactate dehydrogenase (LDH) assay, enzyme-linked immunosorbent assay (ELISA), and oxidative stress while cells were used for MTT assay and cells viability assay.

Cells viability assay

After treatment, cells were treated with crystal violet (Invitrogen Inc., USA) for 20 min. After washing with normal saline, the color taken by cells was solubilized with dimethyl sulfoxide (DMSO) (Invitrogen Inc., USA) and the absorbance of solution was measured at 540 nm.

Cell proliferation assay

To compare the proliferative potential of both groups, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed. The monolayer of cells was first washed with phosphate-buffered saline (PBS) (Invitrogen Inc., USA). 500 μl complete medium, along with 60 μl MTT solution (Invitrogen Inc., USA), was added to cells and incubated for 2 h at 37°C. Purple color crystals that formed within the cells were solubilized with DMSO and its absorbance was taken at 570 nm.

LDH assay

LDH assay was performed using 5 μl medium from each group at the end of treatment using LDH assay kit (AMP Diagnostics, AMEDA Labordiagnostik GmbH, Krenngasse Graz, Austria) according to manufacturer's instructions. Briefly, 5 μl of cell culture medium of both groups was mixed with 95 μl working reagent, incubated for 5 min, and then absorbance was recorded at a wavelength of 340 nm.

Enzyme-linked immunosorbent assay (ELISA)

Solid phase sandwich ELISA was performed for vascular endothelial growth factor (VEGF), p53, and p38. A microtiter plate (Corning, USA) was coated with primary antibodies, i.e., rabbit anti-VEGF (SantaCruz Biotechnology, Finnell St, Dallas, USA), mouse anti-p53, and rabbit anti-p38 (Invitrogen Inc., USA) and incubated for 48 h at 4°C. After washing thrice with tris-buffered saline (TBS), 100 μL cells' supernatant from both groups was loaded and incubated for 18 h. The wells were then washed three times and blocked for 1 h with 10% bovine serum albumin (BSA), leading to overnight incubation with a secondary antibody, i.e., horse raddish peroxidase (HRP) conjugated goat anti-rabbit for VEGF and p38 (SantaCruz Biotechnology, USA) and HRP conjugated goat anti-mouse for p53. After washing, equal volumes of chromogenic solution, 3,3', 5, 5'-tetramethylbenzidine (TMB) (Invitrogen Inc., USA) and 0.1 mM HCl were added to stop the reaction. Using a microtiter plate reader, absorbance was measured at 450 nm keeping 650 nm as reference value.

Estimation of glutathione

The amount of reduced glutathione (GSH) in culture medium was estimated using method of Beutler et al. [11] and Wajid et al. [12] For this, 0.5 mL culture medium from both groups was added in a tube, along with 2.0 mL disodium hydrogen phosphate buffer (0.3 M) and 0.25 ml 5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB (0.001 M) (Invitrogen Inc., USA). The mixture was incubated for 15 min and absorbance was measured at 412 nm.

Estimation of SOD activity

Superoxide dismutase (SOD) activity was assessed by the method of Wajid et al. [12] Briefly, 0.1 mL culture medium was mixed with 1.2 mL sodium pyrophosphate buffer (52 mM, pH 8.3), 0.1 mL phenazine methosulfate (PMS) (186 μM) (SantaCruz Biotechnology, USA), 0.3 mL nitroblue tetrazolium (NBT) (300 μM) (Invitrogen Inc., USA) and reaction was started by the addition of 0.2 mL nicotinamide adenine dinucleotide (NAD) (750 μM) (SantaCruz Biotechnology, USA). After incubation at 30°C for 90 s, the reaction was stopped by the addition of 0.1 mL glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 mL n-butanol. The mixture was incubated for 10 min and centrifuged at 2,000 g for 5 min. The upper butanol layer was taken and its absorbance was recorded at 560 nm.

Estimation of catalase activity

The activity of catalase was monitored by using the method described by Sinha. [13] Culture medium of 0.1 ml was taken and mixed with 1.0 mL phosphate buffer (10 mM, pH 7.0) and 0.4 mL H 2 O 2 (0.2 M) (Sigma Aldrich, USA). The reaction was stopped by adding 2.0 mL dichromate acetic acid reagent. The samples were incubated for 10 min in a boiling water bath, cooled, and the absorbance was measured at 530 nm.

Estimation of malondialdehyde level

Level of malondialdehyde (MDA), a free radical species was evaluated by measuring thiobarbituric acid reactive substances via the method of Wajid et al. and Ohkawa et al. [12],[14] For this, 0.2 mL cell culture medium was added to 0.2 mL sodium dodecyl sulfate (SDS) (8.1%), 1.5 mL thiobarbituric acid (TBA) (0.8%), 1.5 mL acetic acid (20%, pH 3.5) and the volume was made up to 4.0 mL with distilled water and incubated at 90˚C for 60 min. After cooling, 1.0 mL distilled water, 5.0 mL n-butanol-pyridine mixture (15:1) was added and the mixture was shaken vigorously and centrifuged at 4,000 g for 10 min. The upper n-butanol layer was taken and its absorbance was measured at 532 nm.

Statistical analysis

A total of 100 females were enrolled for the study (50 healthy and 50 with breast cancer). The plasma of 10 individuals of each group was pooled. WJMSCs from five samples at passage 3 were provided with each pool of plasma and triplicate of each experiment was performed. Statistical analysis was performed using GraphPad Prism version 5.00 for windows (GraphPad Software, San Diego, California, USA). All results were expressed as mean ± standard deviation (SD). Student's t-test was used for comparison. Statistical significance was considered as P < 0.05.


  Results Top


Effect of plasma from breast cancer patients on viability and growth of WJMSCs

Cell viability was assessed by staining cells with a dye known as crystal violet. It stained all the cells present in a particular well. The color taken up by cells was solubilized and the absorbance of colored solution was compared for both treatment groups. It was observed that cells of Group 1 had more intense color and a higher absorbance value and hence, more viability. It was also observed that cells cultured in cancer plasma showed a significantly lower proliferation compared to the control group as estimated by MTT assay. Cytotoxicity was analyzed by LDH release, which was significantly higher in the medium of Group 2 compared to Group 1 [Figure 1].
Figure 1: Effect of treatment of plasma from breast cancer patients on the growth of WJMSCs (Group 2) as compared to WJMSCs treated with plasma from normal persons (Group 1). (A) Microscopical representation of both groups (200X, scale bar 50 µm) (B) Cells viability assay (C) MTT cells proliferation assay (D) Cell death evaluated by LDH assay. The data are expressed as mean ± SD and P < 0.05 was considered significant for all comparisons. *Denotes significant difference between Group 1 and Group 2

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Gene expression analysis

The expression of VEGF, p53, and p38 genes was evaluated by ELISA. Reduced expression of VEGF while increased expression of p53 and p38 was observed in Group 2 [Figure 2].
Figure 2: Gene expression analysis in WJMSCs treated with plasma from breast cancer patients (Group 2) as compared to WJMSCs treated with plasma from normal persons (Group 1). Sandwich ELISA for (A) VEGF (B) p53 (C) p38. The data are expressed as mean ± SD and P < 0.05 was considered to be significant for all comparisons. *Denotes significant difference between Group 1 and Group 2

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Oxidative stress

Oxidative stress was observed in WJMSCs treated with breast cancer plasma. It was observed that antioxidant enzymes, i.e., the level of GSH, SOD, and catalase activities were significantly low and the MDA level was high in Group 2 compared to Group 1 [Figure 3].
Figure 3: Estimation of oxidative stress in both study groups. (A) Glutathione (B) SOD (C) catalase assay (D) MDA release in medium. The data are expressed as mean ± SD and P < 0.05 was considered to be significant for all comparisons. *Denotes significant difference between Group 1 and Group 2

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  Discussion Top


The current study describes the potential behavior of WJMSCs in breast cancer, which was evaluated by culturing these cells in the plasma from breast cancer patients. These cells at passage 3 were provided with breast cancer patients' plasma and healthy people's plasma in the same percentage, i.e.,10% for 3 days. The rate of cell survival, proliferation, and cell death were estimated by crystal violet staining, MTT assay, and LDH leakage, respectively, while different paracrine factors such as VEGF, p38, and p53 were estimated by ELISA. Oxidative stress and its enzymes were also analyzed in the study.

Cell viability was assessed by staining cells with crystal violet, which is a known dye for the assessment of viability. [15] It was observed that cells cultured in normal plasma were more viable than those cultured in breast cancer plasma [Figure 1]a. MTT after entering the eukaryotic cells was converted into a purple color formazan product in viable cells with active metabolism. [16] MTT cell proliferation assay revealed a decreased rate of cell proliferation in breast cancer-treated cells compared to healthy plasma-treated cells [Figure 1]c. LDH assay was performed on WJMSCs treated with normal plasma and breast cancer plasma. Increased release of LDH in the medium by cells is an indicator of cytotoxicity. [17] LDH release was significantly higher in the medium of cells treated with breast cancer plasma [Figure 1]d.

Angiogenesis is involved in the growth and metastasis of tumors. VEGF was found to be elevated in breast cancer tissue and have a role in tumor angiogenesis through the promotion of proliferation, migration, stabilization, and survival of tumor cells. [18] VEGF was expressed at a reduced level by WJMSCs treated with breast cancer plasma [Figure 2]a. These results indicate the potential positive role of WJMSCs in breast cancer conditions. p38 is an apoptosis-associated protein; similarly, p53 protein is activated by multiple stressors including oncogenic activation and hypoxia. [19] It leads to the activation of genes for apoptosis and cell cycle arrest. [20] Our results showed increased expression of p38 and p53 proteins in Group 2 [Figure 2]b and c, indicating apoptosis of WJMSCs in breast cancer condition.

The generation of excessive free radicals and reduction in endogenous antioxidant defenses lead to oxidative stress conditions. [21] Our results indicate increase in MDA (a free radical species) while significant decrease in GSH, SOD, and catalase (antioxidant enzymes) in Group 2 [Figure 3]. These results show a marked oxidative stress in Group 2.


  Conclusion Top


In conclusion, our study demonstrates that stressed environment of breast cancer induces injury to WJMSCs and reduces their viability. The efficacy of these cells for therapeutics of cancer is reduced in cancer conditions, which necessitates further studies to modulate their survival in breast cancer.

Acknowledgement

The University of Lahore, Lahore, Punjab, Pakistan; INMOL Hospital, Lahore, Punjab, Pakistan.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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