NES1/KLK10 and hNIS gene therapy enhanced iodine-131 internal radiation in PC3 proliferation inhibition

doi:10.1007/s11684-018-0643-y

Front. Med.

2019, Vol. 13

Issue (6) : 646-657 https://doi.org/10.1007/s11684-018-0643-y

RESEARCH ARTICLE

NES1/KLK10 and hNIS gene therapy enhanced iodine-131 internal radiation in PC3 proliferation inhibition

Jiajia Hu¹, Wenbin Shen¹, Qian Qu¹, Xiaochun Fei², Ying Miao¹, Xinyun Huang¹, Jiajun Liu¹, Yingli Wu³(

), Biao Li¹(

)

¹. Department of Nuclear Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². Department of Pathology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
³. Hongqiao International Institute of Medicine, Shanghai Tongren Hospital/Faculty of Basic Medicine, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

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Abstract

NES1 gene is thought to be a tumor-suppressor gene. Our previous study found that overexpression of NES1 gene in PC3 cell line could slow down the tumor proliferation rate, associated with a mild decrease in BCL-2 expression. The BCL-2 decrease could increase the sensitivity of radiotherapy to tumors. Thus, we supposed to have an “enhanced firepower” effect by combining overexpressed NES1 gene therapy and ¹³¹I radiation therapy uptake by overexpressed hNIS protein. We found a weak endogenous expression of hNIS protein in PC3 cells and demonstrated that the low expression of hNIS protein in PC3 cells might be the reason for the low iodine uptake. By overexpressing hNIS in PC3, the radioactive iodine uptake ability was significantly increased. Results of in vitro and in vivo tumor proliferation experiments and ¹⁸F-fluorothymidine (¹⁸F-FLT) micro-positron emission tomography/computed tomography (micro-PET/CT) imaging showed that the combined NES1 gene therapy and ¹³¹I radiation therapy mediated by overexpressed hNIS protein had the best tumor proliferative inhibition effect. Immunohistochemistry showed an obvious decrease of Ki-67 expression and the lowest BCL-2 expression. These data suggest that via inhibition of BCL-2 expression, overexpressed NES1 might enhance the effect of radiation therapy of ¹³¹I uptake in hNIS overexpressed PC3 cells.

Keywords androgen-independent prostate cancer normal epithelial cell-specific 1/kallikrein 10 sodium/iodide symporter radiation therapy proliferation

Corresponding Author(s): Yingli Wu,Biao Li

Just Accepted Date: 20 March 2019 Online First Date: 24 May 2019 Issue Date: 16 December 2019

Cite this article:

Jiajia Hu,Wenbin Shen,Qian Qu, et al. NES1/KLK10 and hNIS gene therapy enhanced iodine-131 internal radiation in PC3 proliferation inhibition[J]. Front. Med., 2019, 13(6): 646-657.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0643-y
https://academic.hep.com.cn/fmd/EN/Y2019/V13/I6/646

Fig.1 Expression of NES1 and hNIS protein in four kinds of PC3 cell lines and verification of function of overexpressed hNIS protein in PC3 cell lines by radioactive iodine uptake related studies in vitro and in vivo. (A) Western blot confirmed the obvious expression of NES1 and hNIS protein in PC3-NES1-hNIS cell line and hNIS protein in PC3-hNIS cell line. Interestingly, weak expression of hNIS protein in PC3-NES1 and PC3-CON cell lines were also detected. (B) The function of overexpressed hNIS protein in PC3-NES1-hNIS and PC3-hNIS cell lines was verified by ¹²⁵I uptake study. Less iodine uptake capacity could be detected in PC3-CON and PC3-NES1 cell lines. (C) In vitro iodine uptake NaClO₄ inhibition study showed that the iodine uptake capacity of PC3-NES1-hNIS and PC3-hNIS cell lines could be completely inhibited by 30 mmol/L NaClO₄. (D) In vitro iodine efflux study showed that Na¹²⁵I was rapidly effluxed from both PC3-NES1-hNIS and PC3-hNIS cell lines, maintaining for approximately 1 h. (E–H) Animal SPECT imaging further confirmed the iodine uptake ability of hNIS protein in vivo. This observation showed that the radioactive iodine uptake of subcutaneous PC3-NES1-hNIS and PC3-hNIS xenografts (red circle and yellow circle) was significantly higher than that of PC3-NES1 and PC3-CON xenografts, respectively (green circle and purple circle). Radioiodine accumulation in PC3-NES1 and PC3-CON xenografts was almost invisible. (I) Quantitative analysis by the automatic g-counter demonstrated a significantly high radioactivity in PC3-hNIS and PC3-NES1-hNIS xenograft tissue compared with that in muscle tissue (17.8-fold and 11.8-fold, P<0.001), a low radioactivity in PC3-NES1, and PC3-CON xenograft tissue compared with that in muscle tissue (3.0-fold respectively, P<0.01). The iodine uptake capacity of PC3-hNIS and PC3-NES1-hNIS xenograft was much higher than that of PC3-CON and PC3-NES1 xenograft (P<0.01), and the uptake capacity of PC3-hNIS was 1.5-fold higher than that of PC3-NES1-hNIS (P<0.05).

Fig.2 Best therapeutic effect of the combined treatment of NES1 and ¹³¹I on PC3 cell lines and xenograft tumors. (A) CCK-8 cell proliferation assay showed an obvious inhibitive effect in the single NES1 gene therapy group PC3-NES1 (P<0.001), single ¹³¹I treatment absorbed by the hNIS protein group PC3-hNIS with ¹³¹I (P<0.01), and the combined treatment group PC3-NES1-hNIS cell line with ¹³¹I (P<0.001). The combined treatment group grew significantly slower than the single NES1 gene treatment group PC3-NES1 cell line (P<0.001) and significantly slower than the PC3-hNIS cell line treated with ¹³¹I (P<0.001). (B) Cell clone formation assay, which lasted for 16 days, showed the most significant proliferative inhibition in PC3-NES1-hNIS cells treated with ¹³¹I. A significant inhibition effect was also found in the PC3-NES1 group and the PC3-hNIS group with single ¹³¹I treatment. The finding was consistent with the result of CCK-8 cell proliferation assay. (C) Quantitative analysis of cell clone formation assay by Image-Pro Plus software showed the same result. Compared with the PC3-CON group, the combined treatment group significantly grew the slowest (P<0.001). A significant inhibitive effect was also observed in the PC3-NES1 group (P<0.001) and the PC3-hNIS group treated with ¹³¹I (P<0.01). Compared with the PC3-NES1 group and the PC3-hNIS group treated with ¹³¹I, the combined treatment group grew significantly the slowest (P<0.001). (D) Tumor volume line graph showed a proliferative inhibition effect on PC3-NES1 and PC3-NES1-hNIS xenograft from the beginning. At days 32 and 46, ¹³¹I systematic therapy was performed twice, with an injection of 37 MBq each. An obvious therapeutic effect could be found in PC3-hNIS-NES1 with the ¹³¹I groups (P<0.01). The growth of PC3-NES1 xenograft was always slower than that of the PC3-CON group (P<0.05). The growth of PC3-hNIS xenograft with ¹³¹I treatment was also slower than that of the PC3-CON group, but the difference was not significant enough. (E) Nude mice body weight line graph showed that the body weight change of each group of nude mice had no obvious significance. However, those loaded with PC3-NES1 and PC3-hNIS-NES1 xenografts increased slightly faster than those loaded with PC3-CON and PC3-hNIS. After day 24, the weight of each group decreased. After ¹³¹I treatment, the weight of PC3-hNIS-NES1 with ¹³¹I group declined the slowest (P<0.01). (F) General images showed that the tumor volume of PC3-hNIS-NES1 with the ¹³¹I groups was the smallest. Single NES1 gene therapy and single ¹³¹I of radiation therapy mediated by the hNIS protein overexpressed groups also had an inhibitive effect compared with the control groups. (G) Tumor inhibition rate (TIR) (mean%±SD%) was as follows: 83.54%±8.79%, 51.91%±6.90%, and 32.46%±17.97% for PC3-CON with PBSvs. PC3-hNIS-NES1 with ¹³¹I, PC3-CON with PBS vs. PC3-NES1 with PBS, PC3-hNIS with PBS vs. PC3-hNIS with ¹³¹I, respectively. Compared with single NES1 gene therapy and single radioactive iodine-131 treatment, combined treatment had the best tumor inhibition rate (P<0.01 and P<0.05, respectively).

Fig.3 Combined therapy of NES1 and ¹³¹I in PC3 xenograft inhibited the tumor proliferation associated with downregulation of BCL-2 expression. (A)¹⁸F-FLT micro-PET/CT imaging, which could evaluate the tumor proliferation in vivo, showed that the combined therapy group (PC3-hNIS-NES1 with ¹³¹I) obviously decreased the volume of xenograft and the ¹⁸F-FLT uptake. The volume of PC3-CON xenograft with and without ¹³¹I treatment was large. The uptake was also high. (B) Bar chart of ¹⁸F-FLT uptake in xenograft showed that the SUV_max of PC3-NES1-hNIS xenograft tumors with ¹³¹I treatment was the lowest in these eight groups. Compared with the PC3-CON group and two single treatment groups, the difference was significant (P<0.01, P <0.05, and P <0.05, respectively). Single radioactive iodine-131 treatment could also decrease the SUV_max of ¹⁸F-FLT uptake in xenograft tissue (P<0.05). Single NES1 gene therapy could decrease the ¹⁸F-FLT uptake, but the difference was not so significant. (C) Immunohistochemistry showed an obvious decrease of Ki-67 expression in two single-treated groups and PC3-NES1-hNIS with the ¹³¹I group. The latter showed the lowest expression. In addition, the BCL-2 expression in the xenograft tissue of both single-treated groups and PC3-NES1-hNIS with the ¹³¹I group decreased obviously. The latter was the lowest.

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