EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: This study sought to observe transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA and examine the combined effect of gemcitabine and siRNA inhibition of telomerase on pancreatic cancer cells.

Materials and Methods: Transfected pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA were selected as target and divided into 9 groups: (1) T1 group (pSilencer4.1-CMV neo-hTERT1-siRNA), (2) T2 group (pSilencer4.1-CMV neo-hTERT2-siRNA), (3) L group (Lipofectamine) (4) M group (mismatch group pSilence4.1-CMV, as negative control), (5) C group (cell group without transfection), (6) blank and gemcitabine group, (7) mismatch siRNA and gemcitabine group, (8) hTERT1-siRNA and gemcitabine group, and (9) hTERT2-siRNA and gemcitabine group. Expression of hTERT mRNA was detected by reverse transcriptase polymerase chain reaction. Viability of cells was measured by colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide assay. Cell cycle and cell apoptosis were measured by flow cytometry. Expression of telomerase protein was measured by Western blot.

Results: Compared with the L group, M group, and C group, expression of hTERT-mRNA and the level of telomerase protein in T1 and T2 group was down-regulated significantly (P < .05), viability of BxPC-3 cells decreased significantly (P < .05), the ratio of cells in G(0)/G(1) stage increased, the ratio of cells in the S stage and the G(2)/M stage decreased, and the ratio of apoptotic cells increased significantly in the T1 and T2 groups. Gemcitabine treatment had a comparable effect. Combination hTERT siRNA and gemcitabine killed twice as many cancer cells, showing a cumulative effect of the treatments.

Conclusions: Transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-CMV neo-hTERT-siRNA represents good RNAi silencing and anti-pancreatic cancer effects in vitro and could potentiate the cytotoxic effect of gemcitabine to pancreatic cancer cells.

Key words : Pancreatic carcinoma, Apoptosis, Anti-tumor therapy RNAi, Telomerase

Pancreatic carcinoma is a dismal disease because of its aggressive biologic phenotype, characterized by an early local invasion, high metastatic potential, late clinical presentation, poor overall prognosis, and high resistance to radiation and chemotherapy. During 2009, about 42 470 individuals were diagnosed with pancreatic cancer and 35 240 in the United States died from the disease.¹ It is the fourth leading death of cancer. The current preferred therapeutic drug to treat pancreatic cancer is gemcitabine, yet the 1-year survival of pancreatic cancer patients treated with gemcitabine is only about 18%, representing a significant but modest advancement in the quality of life.² There clearly is a need for new approaches to treat that.

Telomerase is a ribonucleoprotein complex that includes an RNA template molecule (TER) and a human telomerase reverse transcriptase (hTERT).³ Telomerase is thought to play an essential role in tumorigenesis and progression. The activity of telomerase is directly correlated with the expression of hTERT. Eighty-five percent of all cancers are telomerase positive, while for most normal somatic cells, telomerase activity is absent or very low.^4-6 Therefore, telomerase is considered an ideal target for cancer therapy, and many strategies have been developed to inhibit telomerase, such as antisense nucleotides, ribozymes, dominant-negative proteins, and small synthetic molecules.^{7, 8}

RNA interference (RNAi) represents an evolutionarily cellular mechanism for controlling expression of genes in almost all eukaryotes, including humans.⁹ It is a process of posttranscriptional gene silencing in which double-stranded RNA (dsRNA) target homologous messenger RNA (mRNA) for degradation or blocking mRNA translation. RNAi is initiated when dsRNAs are recognized by Dicer, a member of the RNase III protein family. The Dicer cleaves dsRNAs into short interfering RNAs (siRNAs), which are 21 nucleotides in length and they have 2-nt 3’overhangs.¹⁰ These siRNA duplexes are then incorporated into a protein complex called RNA-induced silencing complex, which recognizes and cleaves the cognate mRNA. Thus, siRNA has now become a powerful tool for studies
on gene function, cancer, and viral disease therapy.

These siRNAs can be produced 4 different ways: chemical synthesis, in vitro transcription, enzymatic digestion of dsRNAs, and transfection of DNA vectors encoding siRNAs or short hairpin RNAs (shRNAs), which can be converted into siRNAs in cells.11 Among the 4 ways, transfection of DNA vectors has some advantages such as low cost, lasting expression of siRNA, and ease of preparation, making it the preferential method for studies.¹²

In this study, we hypothesized that a combination treatment of gemcitabine and hTERT siRNA could sensitize tumor cells to gemcitabine and thus enhance its cytotoxicity. We characterized the effect of our hTERT siRNA in promoting pancreatic cancer cells apoptosis in vitro. We then compared the effects of hTERT siRNA, gemcitabine, and the combined treatment of the 2 to see if a cumulative effect would be observed when the 2 treatments were combined.

Materials and Methods

Cell cultures
Pancreatic cancer cell line BXPC-3 was purchased from Institute of Biochemistry and Cell Biology Shanghai Institute for Biological Science, CAS. The cells were cultured in Dulbecco’s modified eagle’s medium complemented with 10% fetal bovine serum, and incubated at 37°C in a humidified atmosphere with 5% CO2. The medium was changed once in every 3 days, and the cells were transferred into 24-well plates at a density of 3 × 10³ cells/well 1 day before transfection. Cells were divided into 9 groups: (1) T1 group (pSilencer4.1-CMV neo-hTERT1-siRNA), (2) T2 group (pSilencer4.1-CMV neo-hTERT2-siRNA), (3) L group (Lipofectamine), (4) M group (mismatch group pSilence4.1-CMV, as negative control), (5) C group (cell group without transfection), (6) blank and gemcitabine group, (7) mismatch siRNA and gemcitabine group, (8) hTERT1-siRNA and gemcitabine group, and (9) hTERT2-siRNA and gemcitabine group.

Short hairpin RNA preparation and the construction of vector
Plasmid vector pSilencer4.1-CMV neo was purchased from Shanghai Genetimes Technology, Inc. (Shanghai China) (Figure 1). The siRNA target sites were designed by commercial software (Applied Biosystems, CA, USA) according to the sequence of the hTERT. According to the target sites, we designed the dsRNA, which can code the shRNA (Figure 2). ShRNAs targeting hTERT, and a short hairpin RNA (shRNA) negative control were synthesized: shhTERT1, sense 5’- GATCC CACCAAGAAGTTCATCTCC TTCAAGAGA GGAGATGAACTTCTTGGTG AGA -3’, antisense 5’- AGCTTCT CACCAAGAAGTTCATCTCC TCTCTTGAA GGAGATGAACTTCTTGGTG G -3’; shhTERT2, sense 5’- GATCC TCAGACAGCACTTGAAGAG TTCAAGAGA CTCTTCAAGTGCTGTCTGA AGA -3’, antisense 5’- AGCTTCT TCAGACAGCACTTGAAGAG TCTCTTGAA CTCTTCAAGTGCTGTCTGA G -3’; shRNA negative control sense 5’- GATCC ACGTACGTGTATACACGCT TTCAAGAGA AGCGTGTATACACGTACGT AGA -3’, antisense 5’- AGCTTCT TCGTACGTGTATACACGCT TCTCTTGAA AGCGTGTATACACGTACGT G -3’.

Transfection
Transfection reagent LipofectamineTM2000 was purchased from Invitrogen Corporation (Carlsbad, CA, USA). Transfection was performed following the manufacturer’s instructions. Antibiotic selection used G418 (500 µg/mL) was begun 24 hours after transfection and completed in 2 weeks.

Reverse transcriptase polymerase chain reaction
Total RNA was extracted from the cancer cells of groups 1 through 5 forty-eight hours after siRNA transfection using RNA Fast Extraction Kit (BioTeke, Beijing, China) according to the manufacturer’s instructions. Complementary DNA was synthesized using BioTeke super RT kit (BioTeke, Beijing, China). Total RNA (2 µg) was reverse-transcribed by M-MLV transcriptase using Oligo dT primers according to the manufacturer’s instructions. The RT reaction mixture (1 µL) and the polymerase chain reaction (49 µL) mixture was mixed and then amplified with PCR. The hTERT up-stream primer sequence is 5’-CCTGCCGTCTTCACTTCC-3’; hTERT down-stream primer sequence is 5’-TGAACAATGGCGAATCTGG-3’; the β-actin up-stream primer sequence is 5’-CGTATTGGGCGCCTGGTCACCAG-3’; β-actin down-stream primer sequence is 5’-GTCCTTGCCCACAGCCTTGGCAG-3’ (primers from TAKARA Biotechnology [Dalian] Co., LTD.) The conditions for the PCR were 95°C for 5 minutes, 94°C for 40 seconds; 54°C for 1 minute; and 72°C for 3 minutes, for 40 cycles; 72°C for 10 minutes, 4°C for 20 minutes. The RT-PCR products were then electrophoresed on a 1.2% agarose gel containing ethidium bromide. β-actin served as the positive control, and RT-negative tubes served as the negative control.

MTT assay for cell viability
The 3-(4, 5-dimethyl thizol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay was performed to assess the effects of different shRNAs on cell proliferation. At 48 hours after transfection, BxPC-3 cells of groups 1-9 were collected and incubated in 96-well plates (5 × 10³ cells/well) each contained 50 µL of medium. Twenty-four hours after seeding, cells of the appropriate groups were treated with gemcitabine to a final concentration of 100 mM. The rate of cellular proliferation was measured every 24 hours for 96 hours. At the end of each time point, the culture medium was replaced by new medium, and cultured in new medium for 24 hours, 20 µL of 5 mg/mL MTT (Sigma-Aldrich Corp. St. Louis, MO, USA) was added to each well. Four hours later, 150 µL of dimethyl sulfoxide was added to the MTT-treated wells and the absorption at 570 nm was determined by a spectrometer.

Western blotting
Cells were collected 48 hours after transfection. After washing with pre-chilled phosphate-buffered saline, cells were lysed in 1 mL of 1% Nonide P-40, 25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, pH 8.0, containing a 1:50 dilution of a protease inhibitor mixture for 30 minutes on ice. HTERT protein was separated using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrotransferred onto a nitrocellulose membrane and blocked for 3 hours in 3% bovine serum albumin after the transfer. Membranes were incubated overnight at 4°C with an hTERT primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), then washed and incubated with an alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST for 2 hours. Results were quantified by densitometric analysis.

Cell apoptosis and cell cycle analysis
Cells of groups 1 through 9 were collected 48 hours after siRNA transfection. Twenty-four hours after seeding, cells of the appropriate groups were treated with gemcitabine to a final concentration of 100 mM. We used terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay (TUNEL assay) to identify the apoptotic cell. We analyzed cell cycle by flow cytometry using propidium iodide. All procedures were performed following the manufacturer’s instructions.

Statistical analyses
All experiments were performed in triplicate. The t test was used to determine the statistical significance of the data obtained. Values for P < .05 were taken as statistically significant differences between the means.

shRNAs were successfully cloned into plasmids
The shRNAs targeting hTERT and negative control were separately cloned into plasmid Silencer4.1-CMV neo, and the corresponding plasmids were named pSilencer4.1-CMV neo-hTERT1-siRNA (T1), pSilencer4.1-CMV neo-hTERT2-siRNA (T2), and pSilencer4.1-CMV neo-negative control-siRNA. After being digested by BamHI and HindIII, these recombinant vectors yielded 55-bp fragments compared with pSilencer4.1, and this was verified by agarose gel analysis (Figure 3). All constructs were sequence-verified.

shRNAs markedly reduce hTERT mRNA and protein expression levels
Reverse transcriptase polymerase chain reaction experiments showed that hTERT mRNA expression levels were pronounced down-regulation (Figure 4A). The band intensity in correspondence with the cells infected with T1 and T2 became weaker compared with the control and normal cells. T1 and T2 had reduced hTERT mRNA expression levels with respect to cells transfected with control siRNA: 42.2% ± 6.6% (P < .01) and 45.8% ± 6.5% (P < .01). There was no significant difference between them (Figure 4B).

The shRNAs were also successful in knocking down hTERT protein expression. As shown in Figure 5, while the GAPDH internal control showed equal loading among the 5 groups, the level of hTERT protein was noticeably different. The hTERT level of T1- and T2-treated group was markedly lower compared with other groups, and suggests that T1 and T2 could effectively reduce the hTERT protein level.

T1 and T2 inhibited the proliferative potential of pancreatic cancer
Decreased telomerase activity is associated with arrested cell growth; we wonder whether or not the hTERT siRNA-induced reduction in telomerase activity would affect cell viability. The effect of shRNA expression plasmids on BXPC-3 cell proliferation was examined by MTT assay. T1 and T2 significantly decreased the percentage of viable cells in bxpc-3 cell line. The decrease was rapid in group 1 and 2: about 80% cells survived after 24 hours, and at the end of 96 hours, there were only 50% cells alive (Figure 6).⁶

Decrease in cell viability caused by the shRNAs was due to increase in apoptosis and most cells in the G0/G1 phase
To determine the cause of decrease in cell viability by the shRNAs was due to an increase in apoptosis or a decrease in cell proliferation, we checked the amount of apoptotic cells using TUNEL assay. As is shown in Figure 7, the apoptosis rate of BXPC-3 cells transfected with T1 and T2 was 25.9% and 23.3% in the end of the first day; after 96 hours, the apoptosis rate was about 40%, suggesting that T1 and T2 could effectively cause cell apoptosis.

After treating with T1, T2, and gemcitabine, the cells in the G0/G1 phase increased significantly, and the cells in the S phase decreased significantly. Cells in the G2/M phase also decreased significantly (Figure 8).

We suggest the mechanism by which hTERT-shRNA regulates the growth of BXPC-3 cell is by arresting most cells in the G0/G1 phase and decreasing cells in the G2/M and S phase, as well as promoting cell apoptosis.

Combined hTERT siRNA and gemcitabine treatments could additively increase pancreatic cancer cell apoptosis
First, we carried out the MTT assay and TUNEL assay to value the cytotoxicity of gemcitabine alone on BXPC-3 cell lines. The gemcitabine treatment had almost the same cytotoxicity comparable to the hTERTsiRNA treatment; it significantly decreasedthe percentage of viable cells in bxpc-3 cell line (Figure 6). The TUNEL assay analysis showed that the BXPC-3 cells were equally prone to apoptosis induction by either treatment (Figure 7).

The combined use of gemcitabine and hTERT-siRNA showed a cumulative effect. The cells were first transfected with the siRNA, and then gemcitabine was added. From the MTT assay, already half or more than half of the cells did not survive by the end of the first day of the treatments. By the end of day 4, more than twice as many cells were dead from the combined treatment (Figure 6). The TUNEL assay revealed that about 70% cells were apoptotic after 96 hours, which was roughly close to the sum of the apoptotic cells caused by hTERT siRNA and doxorubicin alone, thus again showing an additive effect of the combined treatment (Figure 7).

The use of siRNAs to efficiently induce sequence-specific gene silencing has opened new opportunities in the field of oncology for the validation of new therapeutic targets and the development of innovative anticancer therapies based on the interference with the expression of specific cancer-related genes.¹³ The RNA interference, which is mediated by 19-nucleotide to 23-nucleotide siRNAs, has some shortcomings. The duration of the silencing is short and transferring efficiency is low. That restricts the field of the application of siRNA in mammals. However, the cellular expression of shRNA from vectors will solve all of these shortcomings of siRNA. The shRNAs are expressed from mammalian promoters on DNA vectors, which are introduced to cells by transfection or infection and possess double-stranded stems that are about 19 to 23 nucleotides in length. That can produce long-term, stable, and highly specific gene silencing.

In this study, we used 2 chemically synthesized shRNAs targeting different sequences of hTERT mRNA. Our results have shown that both of our 2 shRNAs could reduce hTERT gene and protein level in Bxpc-3 pancreatic cancer cells. In fact, it has been shown that accessible RNAi target sites may be rare in some human mRNAs,¹⁴ probably as a consequence of local secondary structures, which may limit target accessibility. However, our data indicated that although targeting different sequences, our 2 shRNAs were effective in eliciting the RNAi pathway in pancreatic cancer cells.^15-17

The 2 shRNAs that we designed have succeeded in down-regulating hTERT gene expression and protein level. After treatment with T1 and T2, hTERT gene and protein levels of Bxpc-3 cells were distinctly reduced compared with the control group. We also could observe there are more cells at the stage of G0/G1 and fewer ones at stage S and G2/M in shRNAs-treated groups, which means the declined in cell proliferation vigor. Also, the apoptosis ratio of BxPC-3 in C, M, and L group is comparatively low, while the ratio in T1 and T2 group has obviously increased. Because chromosomes are capped and protect by telomeres, we first speculated that the rapid apoptosis and low cell proliferation vigor was a result of telomere uncapping or shortening. However, Gandellini and associates¹⁸ have shown that the rapid effect by the hTERT siRNA was not due to telomere uncapping or shortening. Recent studies also have shown that telomerase is involved in DNA repair and is an assistant protein that functions in cell viability and proliferation.^{19, 20} So disruption of hTERT expression may impair some other vital cellular functions as well, which may be some reasons for the rapid apoptosis. We wonder if the rapid apoptosis induction by siRNA has some relation with p53 any other DNA repair pathways.

At the same time, in Figure 8, we found that the cell cycle analysis for group 5 showed increased cell cycle progression compared to the group 3 and the group 4. Also, group 5 showed unobvious increased cell growth compared to groups 3 and 4 in Figure 6.

We used Lipofectamine 2000, which has a weak cytotoxic effect to help the transfection. This may affect the proliferation vigor of the cells. So there are more viable cells in group 5 compared to groups 3 and 4.

We also showed that hTERT siRNA could add the cytotoxic effect of gemcitabine. The combined treatment of hTERT siRNA and gemcitabine indeed caused more pancreatic cancer cells deaths than either treatment alone.

Recombinant plasmid Silencer 4.1-CMV neo-hTERT-siRNA could cause degradation targeting BxPC-3 pancreatic cancer cells and down-regulation of hTERT mRNA and telomerase protein expression, and have favorable RNAi silence effect. And demonstrated the potential of inhibiting telomerase as an effective treatment of pancreatic cancer when used alone and, when used in conjunction with gemcitabine, could potentiate the cytotoxic effect of the drug to pancreatic cancer cells.

Volume: 10 Issue: 4 August 2012

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