Journal of Cancer Research and Therapeutics Medknow Publications on behalf of the Association of Radiation Oncologists of India (AROI) ISSN: 0973-1482 EISSN: 1998-4138 Vol. 6, Num. 4, 2010, pp. 478-481

Journal of Cancer Research and Therapeutics, Vol. 6, No. 4, October-December, 2010, pp. 478-481

Original Article

Evaluation of natural killer cell activity in pre and post treated breast cancer patients

Ahmad Piroozmand¹, Zuhair M Hassan²

¹ Department of Microbiology and Immunology, Kashan University of Medical Sciences, Kashan, Iran
² Department of Microbiology and Immunology, Medical Faculty, Tarbiat modarres University, Tehran, Iran

Correspondence Address: Ahmad Piroozmand, Department of Microbiology and Immunology, Kashan University of Medical Sciences, Kashan, Iran, apiroozmand@gmail.com

Code Number: cr10120

PMID: 21358084

DOI: 10.4103/0973-1482.77110

Abstract

Keywords: Breast cancer, flowcytometry, mastectomy, natural killer- Cell

Introduction

Natural killer (NK) cells are the front line defense against tumor cells and act as the main component of immune system to monitor tumors, modified cells, viral infections and some fungal and bacterial infections.

These cells are one of the factors that regulate immune system as well as the function of stem cells which are involved in hematopoiesis. ^{[1],[2],[3],[4],[5],[6],[7],[8]} Thus, the process of proliferation, establishment and metastasizing of cancer is followed by a decrease in NK cell activity. Because of the significant role of NK cells in gaining natural immunity and protection against tumor and infectious agents, we can also examine the quantitative and qualitative function of these cells in blood and other lymphatic organs in order to positively evaluate prognosis and obtain an effective result. ^[9],[10]

In view of various studies in this field that suggest NK cell′s activity decreases in patients suffering from cancer, in particular breast cancer, NK cells could be effective for evaluating the prognosis of treatment. ^[11]

In this study, we evaluated the level and the function of NK cells in patients attended to institute of cancer, Imam Khomeini hospital, Tehran.

Materials and Methods

Patients

Eighteen patients with breast cancer (invasive ductal carcinoma) who underwent surgical procedure were studied in Cancer Institute of ImamKhomeini hospital, Tehran. Patients with a history of chemotherapy and/or radiotherapy were excluded from the study. The mean age of patients was 51.2 ± 8.2 and the range was 3462. Patients were diagnosed pathologically to have invasive ductal carcinoma. Eighteen healthy people were also studied as the control group.

Blood sample

About 10 ml blood was collected from the patients. Peripheral blood lymphocytes were isolated by ficollhypaque, washed thrice with RPMI1640 medium and incubated for 30 min for phagocytes adhering to the plate. ^[12] Effector cells like peripheral blood lymphocytes were counted via trypan blue and their viability was determined. Effector cells RPMI (1X10 ⁵ effector cells/ml) were measured by flowcytometer. The gate of the cell was measured by FLS and PLS.

Target cells

Human erythromyeloblastoid leukemia cell line was cultured in RPMI-1640 medium with 10% FCS. The viability of K562 was measured by trypan blue (around 97% viable) and 100 μl of K562 labeled with 100 μl working solution of PI and measured by flowcytometer. The gate of the cell was measured by FLS and PLS.

Flowcytometric analysis

Natural killer cytotoxicity assay: K562 tumor cell line, was maintained in continuous suspension culture in RPMI 1640 + 10% FCS and harvested during the log phase of growth and used for the evaluation of NK activity. Cells were brought to the concentration of 1 × 10 ⁵ ml. Effector cells were cultured from peripheral blood, washed and brought to a concentration of 5 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ cell/ml in RPMI + 10% FCS. Effector cells and K562 target cells were mixed in E/T ratio of 5:1, 10:1 and 20:1. Briefly, tubes were centrifuged for 7 min at 250 rpm at room temperature to promote conjugate formation and then placed in a water bath at 37°C for 10 min. Conjugates were then gently resuspended in RPMI and then incubated for 1.5 h at 37°C, 5% CO2. A working solution was prepared by adding 0.5 μg/ml of propodium iodide (Sigma) in RPMI 1640 + 10% FCS as previously reported by Vitale et al. ^[13] for staining the DNA of the lysed cells. At the end of the incubation, 100 μl of working solution of PI was added to 100 μl of cells and analyzed by flowcytometry. Target controls were incubated at a concentration of 1 × 10 ⁵ cells/ml in order to monitor the spontaneous death rate. The NK cytotoxicity assay was performed using a FAC Star flowcytometry (Coulter) equipped with an argon ion laser at 200 mW light output. ^[14] FLS and PLS were collected in linear mode.

Lysed target cell

K562 was mixed with 2 ml distilled water for 2 min for complete lyses. Target cell was then centrifuged and resuspended with RPMI, and 100 μl working solution of PI was added to 1 ml of lysed cell and measured by flowcytometer. The gate of the cell was measured by FLS and PLS. First, three distinct populations could be detected on the scatter gram, corresponding to the effector cells, live targets and dead targets. Then effectors were gated out on the basis of PLS, as previously reported, ^[15] and statistical analyses were performed on K562 cluster. The relative amounts of cells with lower FLS and slightly higher PLS values gave the lytic percentage of cells. A total of 10,000 events were collected for each sample. The numbers of cells in the gate of lysed target indicate the quantity of NK cell activity.

Statistical Analysis: One-way analysis of variation (ANOVA) and Kruskal-Wallis nonparametric test were employed using SPSS software.

Results

Evaluation of NK activity in pre and post surgery patients

To evaluate NK cell activity, 18 pre surgical patients were selected and compared with 18 normal people. The results in [Figure - 1] show that the average activity of NK cell in post surgery was significantly (P<0.05) increased compared with pre surgery (37.7% vs 24.4%) patients, which indicates an improvement in the microenvironment of the NK cells. However, NK activity was not recovered to the normal status. A significant difference was seen between NK activity in post surgery and normal NK cell activity in normal healthy individuals (62.5%) [Figure - 1].

Evaluation of NK activity in pre and post surgery in different stages of tumor

NK activity in patients with different stages of tumor was identified as follows: two in stage I, four in stage II, five in stage III and seven in stage IV and the results are given in [Figure - 2]. The activity of NK cell in various stages of disease was different and consequently there is a significant correlation between disease stages and NK cell [Figure - 2]. The NK activity is gradually decreased with the increase in stage of tumor. A significant increase in natural killer activity was noticed in stage I and II post surgery compared to pre surgery.

Correlation between NK activity in pre and post surgery with patient′s ages

To evaluate the correlation between NK activity and the ages of the patients, 18 patients suffering from invasive ductal carcinoma were evaluated; the patients were classified into three main age groups: group 1 (34-46), group 2 (47-53) and group 3(54-62). The results indicated that no relationship exists between NK activity and patient′s ages [Figure - 3].

Discussion

The role of natural killer cells as a factor influencing solid-tumor metastasis has been investigated with great attention. The ability to eliminate circulating tumor emboli is closely associated with the level of host NK. ^{[16],[17],[18],[19]} Low NK results in increased tumor cell survival in the blood stream and metastatic pulmonary lodgment. ^[16]

Promotion of solid-tumor metastasis by surgery is reproducible experimentally; unfortunately, it is also thought to occur occasionally in patients who undergo extirpative solid tumor resection. ^[20] It is important to understand the immunological consequences of surgical intervention in the tumor-bearing host because of its potential role in host defense at this time. Further knowledge of such events may suggest means for immunomodulation in the preoperative period and the possible prevention of subsequent metastases.

NK cells were proved to be of high significance in this monitoring by several studies and there is a general reverse correlation between NK activity and progressing the metastasis of various tumors. ^[4] The assessments show a direct correlation between life span of tumor suppressed patients and NK cell activity. ^[5],[21]

The current study aimed to answer the question of whether there is an increase in NK activity due to tumor volume reduction (post surgery). The results indicated that the NK activity was significantly resumed in patients post surgery.

According to current literature, there is a significant difference between NK cell activity before and after treatment which indicates the efficient role of surgery in decreasing tumor volume and therefore removing the suppressive effect of tumor tissue on the NK cells and increasing their cytotoxicity.

Furthermore, the correlation between tumor stage and NK activity revealed that NK activity significantly decreases with the increase in tumor stages. The current study suggests that there is no correlation between the age and NK cell activity, neither in normal persons nor in individuals experiencing breast cancer. On the other hand, other studies done abroad, ^{[5],[22],[23],[24],[25]} or studies performed by Sheikhi in Iran have shown the NK activity to be 55-70 % in normal individuals based upon Chrome release method which reaches 62.5% using flowcytometry method. ^[21]

As a result, this method is a reliable technique for detecting normal cytotoxicity because of many advantages including reproducibility, accuracy, fast, no risk of radioisotopes and finally feasibility of concurrent qualitative and quantitative assays.

The final conclusion is that mastectomy may cause NK cells to increase their activity in persons suffering from breast cancer and promote the outcome of treatment. ^[26]

Finally, it is recommended that a better understanding of correlation between tumor and NK cell will achieve in light of other therapies (chemotherapy, radiotherapy, and immunotherapy).

In this way, a comprehensive data collection can act as a prognosis factor or it may be used in treatment and delaying disease proliferation.

Acknowledgement

We thank Mr. Montazeri, operator of FACS system, for her excellent assistance. We also thank all cancer center′s nurses in Khomeini hospital for their valuable help in sampling.

References

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