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

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

Introduction

Prostate cancer is the most common andropathy in Euro-American countries. The newest statistical cases of this malignant tumor in the USA in 2017 ranked the first place, accounting for 161 360 and makes up 19% in male malignant cases. Moreover, prostate cancer is the third most lethal malignant tumor after lung and rectum cancers [¹]. The incidence of prostate cancer in China is lower than in Euro-American countries [²]. However, with the aging and Europeanization of dietary and living habits, the disease incidence is rising every year. In addition, the early diagnosis and treatment are lagging behind in developed countries. As a result, the stage of patients in many areas becomes metaphase or reaches the advanced stages with less opportunity provided for radical cure.

Radical cure includes bath radical prostatectomy and radiotherapy [³,⁴], which fits the patients with early and limited prostate cancer [⁵]. The patients with advanced prostate cancer in progressive stage and metastasis are fit for prostatectomy or castrate treatment combined with radiotherapy [⁶]. During the early stage of the therapy, most of the patients have good curative effect, with clinical manifestations of the regression of tumor and level of prostate-specific antigen (PSA) and the relief of symptoms. However, after 12–33 months of androgen deprivation therapy, the patients are gradually converted to androgen independence, or even castration-resistant prostate cancer (CRPC) with very unfavorable prognosis [⁷,⁸]. Chemotherapy and new endocrine therapy drugs, including docetaxel, enulide, and abitron, can only temporarily prolong the survival time of patients with CRPC (2.8–4.8 months) [⁹,¹⁰]. Thus, the focus of this research is to discover effective therapy for prostate cancer.

Increasing studies have found that the development and progression of prostate cancer are associated with the disturbance of different signaling pathways caused by mutations in a variety of different genes, including the inactivation of important tumor-suppressor genes and the activation of oncogenes [¹¹,¹²]. The normal epithelial cell specific-1 (NES1) gene, also known as KLK10, is a member of the kallikrein-related peptidase (KLK) family. NES1 is a secretive type of serine protease and is wildly expressed in breast and prostate tissues [¹³]. In our previous research, we have found that NES1 expression was lost in the androgen-independent prostate cancer cell line PC3. Besides, after the NES1 gene is overexpressed, apoptosis-related gene BCL-2 expression is decreased. We found that the apoptosis of the tumor cells increased, and the proliferation was also inhibited [¹⁴]. This result confirmed the tumor suppressor role of NES1. Protein BCL-2 is an important anti-apoptotic protein, which can inhibit the apoptosis by inhibiting the mitochondrial pathway of apoptosis [¹⁵]. The expression of anti-apoptotic protein BCL-2 was increased remarkably in drug-resistant CRPC; this factor may be one of the causes of prostate cancer resistance [¹⁶].

Single therapy of tumor often does not prevent the occurrence of treatment resistance. Sodium/iodide symporter (NIS) is the trans-membrane glycoprotein of thyroid cells, which can mediate local aggregation of multiple radionuclides, including ¹²⁵I and ¹³¹I; thus, it has been widely applied to the tumor imaging and radiation therapy of different kinds of tumors besides thyroid diseases [¹⁷,¹⁸]. Considering that the BCL-2 downregulation may enhance the effect of radiotherapy [¹⁹,²⁰], this study aims to observe the combined effect on prostate cancer by combining NES1 gene therapy with ¹³¹I radiation therapy absorbed by hNIS protein to increase the effect of ¹³¹I radiation via the downregulated BCL-2 expression by NES1 overexpression.

Materials and methods

Reagents and antibodies

Lentiviral plasmids pLVX-CMV-0-IRES-puro were purchased from Shanghai Boyi Bio-tech Inc. (China). Lentivirus packaging plasmids pVSVG/psPAX2 were also purchased from this company. Puromycin and polyberen were obtained from Sigma-Aldrich, Inc., Merck Company (Germany). Na¹²⁵I and Na¹³¹I reagents were purchased from Shanghai Xinke Pharmaceutical Inc. (China). Cell counting kit 8 (CCK-8) cell proliferation kit was purchased from Kumamoto Dongren Chemical Technology Inc. (Japan). Mouse anti-hNIS monoclonal antibody was purchased from EMD Millipore, Inc., Merck Company (Germany). Rabbit anti-KLK10 and mouse anti-Ki-67 monoclonal antibodies and antirabbit and antimouse horseradish peroxidase (HRP)-labeled secondary antibody were purchased from Sigma-Aldrich, Inc., Merck Company (Germany). HRP-b-actin was purchased from Shanghai Kangchen Bio-tech Inc. (China).

Construction of recombinant lentiviral vectors PC3-NES1-hNIS and PC3-hNIS

Recombinant lentiviral double gene therapy vector pLVX-NES1-IRES-hNIS-puro was designed to transcribe NES1 after the promoter cytomegalovirus (CMV) and to combine hNIS by using an internal ribosomal entry site (IRES). The enzyme restriction sites before and after the gene fragment of NES1-IRES-hNIS were XhoI and BamHI, respectively, as confirmed by gene sequencing. Recombinant lentiviral single gene therapy vector pLVX-hNIS-puro was designed to transcribe hNIS after the promoter CMV. The enzyme restriction site before and after the hNIS gene is BamHI and XbaI, respectively, confirmed by gene sequencing.

Construction of PC3-NES1-hNIS and PC3-hNIS stably transfected cell lines

The recombinant plasmid pLVX-NES1-IRES-hNIS-puro and the two packaging plasmids (pVSVG and pCMVD 8.91) were co-transfected into HEK293T cells by Lipofectamine 2000^TM (Invitrogen^TM/ThermoFisher Scientific Company, USA). After incubation at 37 °C for 48 h, the cell supernatant, which contained NES1-hNIS-puro of the virus solution, was used to transduce NES1 and hNIS genes into PC3 to obtain the NES1 and hNIS overexpressed stable cell line PC3-NES1-IRES-hNIS-puro. Then, 1 mg/mL of puromycin was added for cell screening, which lasted for 2 weeks. Finally, the stably transfected cell line PC3-NES1-hNIS was established. The stable hNIS-overexpressing cell line, named as PC3-hNIS, was obtained by the same method.

Cell lines

Androgen-independent prostate cancer cell line PC3 (ATCC, CRL-1435) was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). HEK293T cell line was provided by Prof. Yingli Wu. PC3-NES1 and PC3-CON cell lines, which were stably transfected with NES1 and blank vector, were successfully constructed in the previous study [¹⁴] and retained in the laboratory. PC3-NES1-hNIS, PC3-hNIS, PC3-NES1, and PC3-CON cell lines were cultured in RPMI1640 medium (Gibco^TM/ThermoFisher Scientific Company, USA) supplemented with 10% fetal bovine serum (FBS, Gibco^TM/ThermoFisher Scientific Company, USA) and 100 units/mL penicillin and 100 mg/mL streptomycin at 37 °C and 5% CO₂. HEK293T cell line was cultured in DMEM with 10% FBS at 37 °C in a 5% CO₂ atmosphere.

Western blot

Cells digested by trypsin were harvested and washed with ice-cold 1× phosphate-bufferred saline (PBS) then were lysed with lysis buffer (62.5 mmol/L Tris–HCl, pH 6.8, 100 mmol/L DTT, 2% SDS, 10% glycerol). Protein extract samples were equally loaded on 10% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred to an immobilon-polyvinylidene fluoride membrane (Schleicher & Schuell, Dassel, Germany). After blocking with 5% nonfat milk in PBS, the membranes were incubated with the following primary antibodies respectively at 4 °C overnight: NES1 (1:1000), hNIS (1:1000), followed by the corresponding HRP-conjugated secondary antibodies (1:2000). HRP-linked b-actin (1:5000) was finally incubated at room temperature for an hour and used as loading control. All experiments were repeated thrice with similar results. Bands were detected by chemiluminescence by using a Pierce ECL Western Blotting Substrate (Thermo Scientific Pierce, USA) and recorded by an Image Quant LAS-4000 Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA). Western blot bands were quantified using ImageJ2 × (2.1.4.7) software and normalized to normalizator signal (b-actin).

In vitro iodine uptake study

PC3-NES1-hNIS, PC3-hNIS, PC3-NES1, and PC3-CON cells (5 × 10⁴ cells/well) were seeded into 24-well plates, incubated at 37 °C in a 5% CO₂ atmosphere overnight. Each well was washed by sterile, ice-cold 1× PBS twice. Then, 0.5 mL of serum-free RPMI1640 culture solution containing 3.7 kBq of Na¹²⁵I and 10 mmol/L of NaI was added to each well and incubated at 37 °C in a 5% CO₂ incubator for 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 10 h, and 26 h. Then, the cells were washed thrice with ice-cold 1× PBS and lysed using 0.5 mL/well of NaOH (1 mol/L) at room temperature. The radioactivity (counts/min, cpm) of the cell lysates was measured by an automatic g-counter (Shanghai Rihuan Company, Shanghai, China), and the time radioactivity curve was plotted. All experiments were repeated thrice with similar results.

In vitro iodine uptake NaClO₄ inhibition study

Sodium perchlorate (NaClO₄, 30 mmol/L) was added into cells before adding serum-free RPMI 1640 solution containing Na¹²⁵I. Other procedures were the same as described above. The radioactivity of the cell lysates was measured, and the time radioactivity curve was plotted. All experiments were repeated thrice with similar results.

In vitro iodine efflux study

Four kinds of cells (5 × 10⁴ cells/well) were incubated with 0.5 mL of serum-free RPMI1640 culture solution containing 3.7 kBq of Na¹²⁵I and 10 mmol/L of NaI at 37 °C in a 5% CO₂ incubator for 1 h, which was the uptake pick according to the result of iodine uptake study. Other procedures were similar to that of iodine uptake study. Then, cells were washed thrice with ice-cold 1× PBS and incubated with 0.5 mL of serum-free RPMI 1640 solution containing 10 mmol/L NaI (without radioactive Na¹²⁵I) at 37 °C in 5% CO₂ incubator for 5, 10, 20, 30, 60, and 120 min. The supernatants were removed into the measuring tube and recorded as supernatants. The cells were lysed using 0.5 mL/well of NaOH (1 mol/L) at room temperature and recorded as intracellular specimens. The radioactivity of supernatants and intracellular specimens were measured, and the time radioactivity curve was plotted as described above. All experiments were repeated thrice with similar results.

CCK-8 cell proliferation assay

CCK-8 cell proliferation assay was performed to detect cell activity difference after ¹³¹I radiation therapy in vitro. PC3-NES1-hNIS, PC3-hNIS, PC3-NES1, and PC3-CON cells (4 × 10⁵ cells/well) were seeded into 6-well flat plates and incubated overnight. Each well was washed by sterile, ice-cold 1× PBS twice. Then, 1 mL of serum-free RPMI 1640 culture solution containing 3.7 MBq of Na¹³¹I and 10 mmol/L of NaI was added to each well and incubated at 37 °C in a 5% CO₂ incubator for 16 h. The same volume of 1× PBS and 10 mmol/L NaI serum-free RPMI1640 culture solution was used as a negative control. Then, each well was washed thrice with sterile, ice-cold 1× PBS. After digestion with trypsin, these four kinds of cells after Na¹³¹I treatment were seeded into a 96-well plate (5 × 10³ cells/well) and incubated for 4 consecutive days at 37 °C in 5% CO₂ atmosphere. To avoid the influence of Na¹³¹I, PC3-NES1-hNIS, PC3-hNIS, PC3-NES1, and PC3-CON cells without Na¹³¹I treatment were seeded into another 96-well plate (5 × 10³ cells/well) as control and incubated at 37 °C in another 5% CO₂ incubator for 4 consecutive days. Cellular proliferation was assessed at the moment of cell attachment at approximately 7 h after cells were plated and then at 24, 48, 72, and 96 h by using a CCK-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. After being cocultured for 2 h, the absorbance was detected at a wavelength of 450 nm and adjusted at a wavelength of 690 nm. Each experiment was conducted in triplicate and repeated thrice.

Cell clone formation assay

Cell clone formation assay was also performed to detect cell activity difference after ¹³¹I radiation therapy in vitro. After 16 h treatment of Na¹³¹I (3.7 MBq) at 37 °C in a 5% CO₂ incubator, these four kinds of cells were washed thrice with sterile, ice-cold 1× PBS. After digestion with trypsin, cells were seeded into 6-well plates (1 × 10³ cells/well) and cultured with 3 mL fresh complete culture medium. Four kinds of cells treated by 1× PBS as negative control were also seeded into 6-well plates (1 × 10³ cells/well) and cultured with 3 mL fresh complete culture medium in another 5% CO₂ incubator at 37 °C to avoid the Na¹³¹I influence from the treatment groups. The medium in each well was replaced every 4 days. After 16 days’ cell culture, each well was washed thrice with ice-cold 1× PBS, fixed by 4% weight/volume (w/v) paraformaldehyde for 30 min and stained by 1% Crystal Violet Staining Solution (Beyotime Inst Biotech) for 2 h. Each experiment was conducted in triplicate and repeated thrice.

Establishment of xenograft tumors in nude mice

Experiments were performed on male nude mice of 4 weeks old purchased from the Experimental Animal Center, Shanghai Jiao Tong University School of Medicine. All animal experiment protocols were performed in accordance with relevant guidelines and approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). Target cells were suspended in sterile, ice-cold 1× PBS, and 5 × 10⁶ cells (0.1 mL) were injected into the subcutaneous part of each flank of nude mice with PC3-NES1 or PC3-CON cell suspension on the left side and PC3-NES1-hNIS or PC3-hNIS cell suspension on the right side. The mouse model groups were as follows: the PC3-NES1 (left) and PC3-NES1-hNIS (right) nude mouse model and PC3-CON (left) and PC3-hNIS (right) nude mice model, each with nine mice. Every 4 days, the vital signs were examined, and the mice weight and the tumor size were recorded, including the long (a) and short (b) diameters of tumor. The volume formula is as follows: volume [mm³] = ab²/2. When the tumor grew large enough (long diameter was up to 1.5 cm) and the weight of each group decreased, three of the PC3-NES1 (left) and PC3-NES1-hNIS (right) nude mice and PC3-CON (left) and PC3-hNIS (right) nude mice were used for ¹³¹I in vivo imaging. Three of those mice were used for ¹³¹I in vivo treatment, and the last three were injected with sterile 1× PBS as negative control treatment.

In vivo ¹³¹I single-photon emission computed tomography (SPECT) imaging

To detect the iodide uptake ability of hNIS overexpressed PC3 cells in vivo, three of the PC3-NES1 (left) and PC3-NES1-hNIS (right) nude mice and PC3-CON (left) and PC3-hNIS (right) nude mice were injected with 3.7 MBq of Na¹³¹I through tail vein for each one. Gasification anesthesia was administered and maintained by volume fraction of 3% isoflurane. Mice were spread in prone position and scanned at 30 min after injection by using pinhole collimator static SPECT imaging (Infinia, GE Company, USA). The image acquisition magnification was 1.45, and the matrix was 128 × 128. SPECT acquisition collected 10⁶ counts per projection, and the scan time was 8–10 min. The images were reconstructed using Xeleris software. Then, the mice were sacrificed by cervical dislocation, and the tumors were taken out. The radioactivity (counts/min, cpm) of the tumor tissue was measured by an automatic g-counter (Shanghai Rihuan Company, Shanghai, China).

In vivo ¹³¹I therapy

At the first 2 weeks of the xenograft tumor formation, L-thyroxine at a concentration of 5 mg/L was added to the drinking water to maximize ¹³¹I uptake by the tumors and to inhibit the uptake by the thyroid gland. Treatment was started when the tumor-long diameter was up to approximately 1.5 cm and the weight of each group decreased. One group (three for each group) of PC3-NES1 (left) and PC3-NES1-hNIS (right) and one group of PC3-CON (left) and PC3-hNIS (right) nude mice were injected with Na¹³¹I intraperitoneally twice on days 1 and 14 with 37 MBq (0.2 mL) for each one and each time. The last groups (three for each group) were injected with 0.5 mL sterile 1× PBS in the same manner as negative control treatment. The vital signs of nude mice were observed every 2 days. The mice weight and the tumor size, with long and short diameter, were measured every 7 days after first ¹³¹I injection until day 28 by using calipers. At day 29, ¹⁸F-fluorothymidine (¹⁸F-FLT) micro-positron emission tomography/computed tomography (micro-PET/CT) imaging was performed. The tumor growth curve was drawn with the tumor volume. The tumor inhibition rate (TIR) was calculated as follows: TIR= (Vc − Vt) / Vc × 100%, where Vc is the mean volume of transplanted tumors in each control group and Vt is the mean volume of transplanted tumors in each ¹³¹I treatment group. All nude mice were sacrificed after ¹⁸F-FLT micro-PET/CT imaging. The transplanted tumor was obtained to compare the size and to undergo immunohistochemical analysis.

¹⁸F-FLT micro-PET/CT imaging

All mice treated by ¹³¹I therapy and sterile 1× PBS underwent ¹⁸F-FLT micro-PET/CT imaging on the Siemens Inveon Micro PET/CT (Inveon MM Platform, Siemens Preclinical Solutions, Knoxville, Tennessee, USA) with a computer-controlled bed and 8.5 cm transaxial and 5.7 cm axial fields of view. The imaging method was based on previous studies [¹⁴]. Mice were anesthetized with 3% volume/volume (v/v) isoflurane in O₂ gas for ¹⁸F-FLT tail intravenous injection (a single injection of 0.2 mL at an activity of 5.3–6.4 MBq). Waiting for 30 min post injection, mice were first computed tomography (CT) scanned for 20 min and then continuously positron emission tomography (PET) scanned for 5 min. Mice were anesthetized with 1.5% v/v isoflurane during scanning. Inveon Acquisition Workplace (IAW) was used for scanning. The CT data were used for both scatter and attenuation correction. Images were reconstructed by an OSEM3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm followed by Maximization/Maximum a Posteriori (MAP) or Fast MAP provided by IAW. The 3D regions of interest were drawn over the tumor tissue guided by CT images, and tracer uptake was measured using the software of Inveon Research Workplace 3.0. Individual quantification of the ¹⁸F-FLT uptake in each of them was calculated to obtain the SUVmax.

Immunohistochemistry

After the ¹⁸F-FLT micro PET/CT imaging of each group, the mice were sacrificed by cervical dislocation, and the tumors were harvested. Then, the tumor tissue was bar cut into almost 1 mm³, immobilized in 4% w/v paraformaldehyde, embedded, and chipped. Tumor tissue sections were prepared into a 4 µm section from paraffin-embedded block and then subjected to immunohistochemical analysis by using mouse anti-hNIS antibody (1:200), rabbit anti-KLK10 antibody (1:200), and mouse anti-Ki-67 antibody (1:200). The experimental method was based on previous studies [¹⁴].

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 software. The comparison of mean of each group was analyzed by one-way ANOVA. Statistical comparisons between the two groups were performed using Student’s t tests. A value of P<0.05 (*) was considered statistically significant, P<0.01(**) and P<0.001(***) were considered remarkably significant, and P>0.05 (ns) was considered no difference. Data were presented as the mean with standard deviation (SD) in the line or bar graphs.

Results

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

The results of Western blot (Fig. 1A) showed that the expression of NES1 and hNIS protein in PC3-NES1-hNIS cell line and hNIS protein in PC3-hNIS cell line were obvious in accordance with the expectation. Interestingly, weak expression of hNIS protein in PC3-NES1 and PC3-CON cell lines were also detected. This finding suggested the endogenous expression of hNIS protein in PC3 cell line. However, less iodine uptake capacity could be detected in PC3-CON and PC3-NES1 cell lines by in vitro ¹²⁵I uptake-related studies (Fig. 1B). The function of overexpressed hNIS protein in PC3-NES1-hNIS and PC3-hNIS cell lines was obvious. In ¹²⁵I uptake study, the peak of iodine uptake in PC3-NES1-hNIS cell line occurred at 2 h with a slow rise stage between 30 min and 2 h, and the peak value was 4.6-fold higher than that of PC3-NES1 and PC3-CON cell lines at the same time point. After 2 h, the uptake value decreased gradually and slowly. However, the iodine capacity was still three-fold higher than that of control cell lines at 26 h. In PC3-hNIS cell line, because of the liaison of hNIS gene directly behind of CMV promoter, the peak value of iodine uptake was higher than that of PC3-NES1-hNIS cell line, with 6.2-fold higher than that of control cell lines. However, the peak time of PC3-hNIS cell line occurred early at 1 h without obvious slow rise stage. After 1 h, the uptake value decreased gradually, and the iodine capacity was 3.1-fold higher than that of control cell lines at 26 h (Fig. 1B). The iodine uptake capacity of both cell lines could be completely inhibited by 30 mmol/L NaClO₄ (Fig. 1C). Given that the internalized iodide cannot be organified as the organic iodide polymer and neither can it be stored in PC3-NES1-hNIS and PC3-hNIS cells like in thyroid cells, Na¹²⁵I was rapidly effluxed from both cell lines, maintaining for approximately 1 h (Fig. 1D).

Animal SPECT imaging further confirmed the iodine uptake ability of hNIS protein in vivo (Fig. 1E–1H). After 30 min of Na¹³¹I injection (3.7 MBq/mouse) in the tail vein of the nude mice, SPECT imaging showed the radioactive iodine uptake of subcutaneous PC3-NES1-hNIS and PC3-hNIS xenografts (red and yellow circles), which were significantly higher than PC3-NES1 and PC3-CON xenografts, respectively (green and purple circles). Significant radioiodine accumulation was also observed in tissues which expressed endogenous NIS, including the thyroid and stomach. Although PC3-NES1 and PC3-CON cell lines expressed slightly endogenous NIS protein, radioiodine accumulation in these two xenografts was almost invisible. To avoid the uptake influence from other organs, we removed the xenografts to detect the radioactivity (Fig. 1I). Quantitative analysis by the automatic g-counter demonstrated a significantly high radioactivity in PC3-hNIS and PC3-NES1-hNIS xenograft tissues (17.8-fold and 11.8-fold, P<0.001), a low radioactivity in PC3-NES1, and PC3-CON xenograft tissue (3.0-fold respectively, P<0.01) compared with the muscle tissue. The iodine uptake capacity of PC3-hNIS xenograft was 1.5-fold higher than that of PC3-NES1-hNIS (P<0.05). This finding was consistent with the radioiodine uptake study result in vitro.

Cell proliferation was reduced significantly by combined treatment of NES1 and ¹³¹I

Four groups of PC3 cell lines (4 × 10⁵ cells/well) were treated with ¹³¹I (3.7 Mq/well) for 16 h, and as control, other four groups were treated with equal volume of 1× PBS at the same time. Then, those eight group cell lines were performed CCK-8 cell proliferation assay for 4 consecutive days. The result showed an obvious inhibitive effect in the single NES1 gene therapy group PC3-NES1, single ¹³¹I treatment absorbed by the hNIS protein group PC3-hNIS with ¹³¹I, and the combined treatment group PC3-NES1-hNIS cell line with ¹³¹I (Fig. 2A). As we expected, the combined treatment group grew the slowest. The combined treatment group grew significantly slower than the single NES1 gene treatment group PC3-NES1 cell line (P<0.001) and was significantly slower than the PC3-hNIS cell line treated with ¹³¹I (P<0.001). The single NES1 gene therapy group PC3-NES1 and the single ¹³¹I treatment group PC3-hNIS cell line with ¹³¹I also had an inhibitive effect compared with the control group (P<0.001 and P<0.01, respectively). However, these cell lines were still growing in the first few days after ¹³¹I treatment. The inhibitive effect was much more intuitive and significant in cell clone formation assay, which lasted for 16 days. This result showed the most significant inhibition proliferative effect in PC3-NES1-hNIS cells treated with ¹³¹I (Fig. 2B). Slow proliferation 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. Quantitative analysis by Image-Pro Plus software showed the same result (Fig. 2C). Compared with the PC3-CON group, the combined treatment group grew significantly 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).

Best therapeutic effect of combined treatment of NES1 and ¹³¹I in PC3 xenograft tumors expressing NES1 and hNIS in vivo

Tumor volume line graph showed a proliferative inhibition effect on PC3-NES1 and PC3-NES1-hNIS xenograft from the beginning (Fig. 2D). The effect was significant since day 24 compared with the PC3-CON and PC3-hNIS groups (P<0.05). The weight change of each group of nude mice had no obvious significance, whereas the groups loaded with PC3-NES1 and PC3-hNIS-NES1 xenografts increased slightly faster than those loaded with PC3-CON and PC3-hNIS (Fig. 2E). After day 24, the weight of each group declined. At day 32, when the tumor long diameter was up to approximately 1.5 cm, we started the radioactive iodine-131 treatment, with an injection of 37 MBq per mouse. Without a significant inhibitive effect, at day 46, we performed the second radioactive iodine-131 treatment with the same radioactivity. After two times radiation treatment, the tumor volume of PC3-hNIS-NES1 and PC3-hNIS with the ¹³¹I groups grew slowly and started to decline. 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 treated was also slower than that of the PC3-CON group, but the difference was not significant (Fig. 2D and 2F). After ¹³¹I treatment, the weight of PC3-hNIS-NES1 in the ¹³¹I group declined the slowest (P<0.01, Fig. 2E). This result showed that combined therapy did not affect food intake or physical activity. By comparing the tumor volume difference between treatment and the control groups, TIR (mean%±SD%) was as follows: 83.54%±8.79%, 51.91%±6.90%, and 32.46%±17.97% for PC3-hNIS-NES1 with ¹³¹I and PC3-NES1 and PC3-hNIS with ¹³¹I, respectively (Fig. 3G). Compared with single NES1 gene therapy and single radioactive iodine-131 treatment, combined treatment had the best TIR (P<0.01 and P<0.05, respectively).

Combined treatment of NES1 and ¹³¹I in PC3 xenograft tumors inhibited the tumor proliferation associated with the downregulation of BCL-2 expression

Before sacrificing the nude mice, we performed ¹⁸F-FLT micro-PET/CT imaging to evaluate the tumor proliferation in vivo (Fig. 3A). The combined therapy group (PC3-hNIS-NES1 with ¹³¹I) 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 (Fig. 3B). 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, whereas the difference was not so significant (Fig. 3B). Immunohistochemistry showed an obvious decrease of Ki-67 expression in two single-treated groups and PC3-NES1-hNIS with the ¹³¹I group. The latter had the lowest expression (Fig. 3C). This result was consistent with all other results of experiments in vivo. Based on the previous study [¹⁴], we detected that the BCL-2 expression in xenograft tissue with combined therapy was the lowest. In addition, both single NES1 gene therapy and single radioactive iodine-131 treatment could decrease the BCL-2 expression (Fig. 3C).

Discussion

NES1 gene is thought to be a tumor-suppressor gene, which decreased the expression in prostate cancer tissues but is expressed in the normal prostate tissues [²¹]. The lack of NES1 expression is due to the hypermethylation of its exon 3 CpG island [²²]. The anticancer effect was first discovered in related studies about NES1 gene therapy of breast cancer, showing that overexpression of NES1 gene or demethylation of its exon3 CpG island could inhibit tumor proliferation [²¹]. Our previous study [¹⁴] and this study found that overexpression of NES1 gene in PC3 cell line could slow down the proliferation rate of PC3 cells and its xenograft tumor. However, the tumor volume was still increasing. These results indicated that the desired effect of tumor inhibition was not easy to be achieved by single gene therapy. Similar to many current cancer treatment programs, single gene therapy is no longer able to be the only treatment regimen. Thus, to combine chemotherapy or radiotherapy on the basis of gene therapy to obtain a possible radical cure is necessary [²³].

Prostate cancer is one of the most popular types of neoplasms treated by radioactive iodine therapy mediated by hNIS gene. From the earliest works about hNIS gene transported by simple adenovirus vector into tumor cell to mediate ¹³¹I therapy in vivo [²⁴] to late ones utilizing tumor-targeted oncolytic virus as vector [¹⁷,²⁵], the tumor inhibition effect of this method has been confirmed by many preclinical studies. Moreover, some researchers have conducted phase I clinical trials to confirm the feasibility of this “radiovirus treatment” [¹⁸]. The results showed that tumor cells could be killed in patients with localized prostate cancer. The advantage of hNIS gene is that it could not only be used for the report gene imaging to monitor the expression reliability of targeted gene and diagnostic localization of tumor but also be used for the radionuclide therapy by ¹³¹I uptake. This phenomenon suggested that by inducing the expression of hNIS protein uptaking ¹³¹I in prostate cancer, ¹³¹I internal radiation therapy would be beneficial to patients, especially those with systemic metastasis of prostate cancer. Given that this treatment does not depend on the dependence of the tumor on androgen, this therapy might be an effective treatment for patients with CRPC.

However, single radiation therapy is also not enough for prostate cancer treatment. This study showed that ¹³¹I treatment could slow down the tumor growth, but the effect was not good enough. The TIR was only 32.46%±17.97%. Our previous study found that NES1 overexpression was associated with a mild decrease in BCL-2 expression in PC3 cells [¹⁴]. According to some literatures, the decrease in BCL-2 expression could increase the sensitivity of radiotherapy to tumors [¹⁹,²⁰]. We supposed to have an “enhanced firepower” effect by combining NES1 gene therapy and ¹³¹I radiation therapy uptake by hNIS protein. Thus, we performed those related functional experiments to evaluate the combined therapy effect on prostate cancer proliferation by using PC3-NES1-hNIS stably transfected cell line.

In this study, we co-expressed NES1 and hNIS genes in PC3 cell line and confirmed the expression by Western blot. Interestingly, we found light endogenous expression of hNIS protein in PC3-NES1 and PC3-CON cell lines. However, this endogenous protein had much less radioactive iodine uptake function in those cells than hNIS-overexpressed cell lines PC3-NES1-hNIS and PC3-hNIS. The animal SPECT imaging also confirmed the function of hNIS-overexpressed cell lines PC3-NES1-hNIS and PC3-hNIS xenograft in vivo, thereby showing that radioactive iodine uptake imaging in xenograft tissues of PC3-NES1 and PC3-CON was not obvious. We wondered whether the endogenous hNIS protein in PC3 cell line lost its iodine uptake function. Quantitative analysis by the automatic g-counter demonstrated a low radioactive iodine uptake in PC3-NES1 and PC3-CON xenograft tissues, i.e., 3.0-fold of that of muscle tissue. Low expression of hNIS protein in PC3 cell line might have led to low iodine uptake. Overexpression of hNIS protein in PC3 cell line could enhance the iodine uptake in PC3 cell line.

Through in vitro and in vivo tumor proliferation studies and the ¹⁸F-FLT Micro-PET/CT imaging, combining NES1 gene therapy with ¹³¹I radiation therapy uptake by hNIS protein overexpressed had a best tumor proliferative inhibition effect. This therapy could decrease the growth line of tumor rather than simply slowing down the growth rate. Immunohistochemistry showed a significant decrease of Ki-67 expression in PC3-NES1-hNIS xenograft tumor treated with ¹³¹I. This finding confirmed the results of those functional experiments. In addition, the weight of this group declined the slowest. This result showed that combined therapy did not affect food intake or physical activity and even gave a good life quality.

In this study, immunohistochemistry showed that both single NES1 gene therapy and single radioactive iodine-131 treatment could decrease the BCL-2 expression. This method could be the reason for tumor proliferation inhibition. Zhao et al. confirmed the role of ¹³¹I in cell proliferation in a thyroid cancer cell line by inhibition of BCL-2 expression in a dose-dependent manner and by upregulation of B cell translocation gene 2-mediated activation of JNK/NF-kB pathways [²⁶]. In addition, the BCL-2 expression in xenograft tissue with combined therapy was the lowest. The treatment effect was also the best. This finding suggested that overexpressed NES1 via inhibition of the BCL-2 expression could enhance the effect of ¹³¹I radiation. The exact underlying mechanism of BCL-2 downregulation is unknown. In our previous study [¹⁴], NES1/KLK10 upregulation decreases BCL-2 expression and Hexokinase2 (HK2) in PC3 cells. Moreover, HK2 downregulation could reduce BCL-2 protein. Thus, KLK10 could decrease BCL-2 through HK2 downregulation. Further molecular mechanism should be explored in future experiments.

Gene therapy for prostate cancer has been used for clinical trials [²³] for those patients with locally recurrent prostate cancer or progressive diseases, such as CRPC. However, manipulation of two target genes still has its limitations, including targeting and expression, especially in relation to the transport vector. In our study, we used lentiviral plasmids pLVX-CMV-0-IRES-puro as vector and used IRES to separate NES1 and hNIS gene to express them, respectively, and to avoid the formation of fusion protein. The protein expression and function results were promising. The targeting will be solved in the future.

In summary, although this study was similar to the pattern of basic studies about gene therapy combined with internal radiation therapy, this work was the first time to combine NES1 and hNIS gene therapy with ¹³¹I internal radiation therapy mediated by hNIS overexpressed for androgen-independent prostate cancer. The combined treatment had efficacy, and a promising result was achieved. PC3 cells had a weak hNIS expression, and overexpression of hNIS protein in PC3 cell line could enhance the cell iodine uptake. BCL-2 downregulation might be the key point of the good combined effect. Further molecular mechanism should be explored in future experiments. Besides, to perform transformation studies or even clinical trials carried out for systematic treatment, a suitable transport vector is needed, such as a safe-targeted virus or a safe nanocarrier. Furthermore, regarding the dose of ¹³¹I systematic therapy, the present study found a significant therapeutic effect in the short-term (4 weeks) after treatment with the therapeutic schedule of twice injection of 37 MBq each at an interval of 2 weeks. The next step is to study whether a combination therapy involving a small dose of ¹³¹I also has the same antitumor effect and the time of onset. These results could be an experimental basis for future clinical application.

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