Study on the Killing Effect of γδT Cells Activated by Rukangyin on Breast Cancer MDA-MB-231 Cells
Authors: Chen Zaiwang , Zhao Yujuan , Li Xiangqi , Wang Keqiang 
Published: October 2021

Objective. To investigate the killing effect of rukangyin (RKY) activated γδT cells on breast cancer cells MDA-MB-231 and to provide a basis for Chinese medicine combined with immunotherapy for breast cancer. Methods. Thus, study isolates peripheral blood mononuclear cells (PBMC) and uses CCK8 to select the optimal concentration of Rukang drink, ZOL (zoledronic acid), and PHA (phytoagglutinin) to activate γδT cells. There are 8 groups including the ① PBMC control group, ② RKY group, ③ ZOL group, ④ PHA group, ⑤RKY+ZOL group, ⑥RKY+PHA group, ⑦ZOL+PHA group, and ⑧ RKY+ZOL+PHA group. At 0 and 14 days of culture, cell viability and γδT cell expansion were detected by flow cytometry. The 8 groups of amplified γδT were cocultured with breast cancer MDA-MB-231 cells labeled with fluorescent dye CFSE at a ratio of 10 : 1 to determine the lethality of γδT cells on breast cancer MDA-MB-231 cells. Results. The optimal concentrations of RKY, ZOL, and PHA to activate γδT cell proliferation were 4.5 mg/l, 3 μM, and 60 μg/ml, respectively. On day 0 of culture, the values (

x
¯

±
s

, %) of γδT cells in groups ① to ⑧ were

3.50
±
0.72

,

3.97
±
0.45

,

3.99
±
0.15

,

4.37
±
0.24

,

4.47
±
0.97

,

4.59
±
1.35

,

3.45
±
0.40

, and

3.89
±
0.48

, while when a comparison between groups was made,

F
=
1.093

and

p
=
0.412

; there is no significant difference between groups. Besides, when being cultured for 14 days, the values (

x
¯

±
s

, %) of γδT cells in groups ① to ⑧ were

4.77
±
0.78

,

23.22
±
2.73

,

26.4
±
0.92

,

28.66
±
1.43

,

27.99
±
1.10

,

30.21
±
1.91

,

32.51
±
0.74

, and

33.21
±
0.42

. Then, based on the comparison between groups,

F
=
119.917

and

p
<
0.001

, there are obvious statistical differences between groups. Furthermore, the expansion values of γδT cells were compared before and after culture for 0 and 14 days. The

t

values of group ① to group ⑧ were 2.072, 12.051, 41.641, 29.015, 27.777, 18.972, 59.836, and 79.622. Except for the PBMC control group (

p
=
0.107

), there are significant statistical differences (

p
<
0.001

). The number of γδT cell expansion at 14 days was the RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>PHA group>RKY+ZOL group>ZOL group>RKY group>PBMC control group. From group ① to group ⑧, the γδT cell expansion multiples were

1.14
±
0.44

,

5.25
±
0.77

,

5.70
±
0.89

,

6.05
±
1.03

,

6.21
±
0.09

,

6.76
±
1.46

,

7.52
±
1.05

, and

7.97
±
1.55

, respectively, while the comparison between groups was

F
=
17.772

and

p
<
0.001

. As for the amplification factor, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY+ZOL group>PHA group>ZOL group>RKY group>PBMC control group. In the killing experiment, the killing rate (

x
¯

±
s

, %) of group ① to group ⑧ was

1.08
±
0.03

,

1.89
±
0.14

,

1.22
±
0.11

,

1.31
±
0.09

,

1.48
±
0.10

,

2.02
±
0.21

,

2.18
±
0.27

, and

2.37
±
0.35

, whereas the comparison between groups was

F
=
20.498

and

p
<
0.001

. In terms of killing rate, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY group>RKY+ZOL group>PHA group>ZOL group>PBMC control group. Conclusion. Rukangyin can increase the lethality of γδT cells against MDA-MB-231 cells by activating the proliferation of γδT cells, which provides a basis for Chinese medicine combined with immunotherapy for breast cancer.