K-ras oncogene silencing strategy reduces tumor growth and enhances gemcitabine chemotherapy efficacy for pancreatic cancer treatment

Abstract

Pancreatic adenocarcinoma remains a fatal disease characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. Gemcitabine is the current standard chemotherapy for advanced pancreatic cancer, but it is still far from optimal and novel therapeutic strategies are needed urgently. Mutations in the k-ras gene have been found in more than 90% of pancreatic cancers and are believed to play a key role in this malignancy. Thus, the goal of this study was to investigate the impact of k-ras oncogene silencing on pancreatic tumor growth. Additionally, we examined whether combining k-ras small interfering RNA (siRNA) with gemcitabine has therapeutic potential for pancreatic cancer. The treatment of tumor cell cultures with the corresponding k-ras siRNA resulted in a significant inhibition of k-ras endogenous expression and cell proliferation. In vivo, tumor xenografts were significantly reduced with k-ras siRNA(GAT) delivered by electroporation. Moreover, combined treatment with pSsik-ras(GAT) plus gemcitabine resulted in strong growth inhibition of orthotopic pancreatic tumors. Survival rate was significantly prolonged and the mean tumor volume was dramatically reduced in mice receiving the combined treatment compared with single agents. Collectively, these findings show that targeting mutant k-ras through specific siRNA might be effective for k-ras oncogene silencing and tumor growth inhibition. The improvement of gemcitabine-based chemotherapy suggests that this strategy might be used therapeutically against human pancreatic cancer to potentiate the effects of conventional therapy.

Figure 1

Schematic diagram showing the structure of the short hairpin RNA pSsik‐ras vector (pSsik‐ras). The different sequences, corresponding to wild type and that containing mutated codon 12, were annealed and ligated into the pSUPER vector.

Figure 2

Determination of the optimal conditions for small interfering RNA (siRNA) transfection. (a) After 24 h of cell culture, specific green fluorescent protein (GFP) siRNA was cotransfected with the EGFP‐N1 plasmid using different concentrations of GFP siRNA and the plasmid expressing GFP. (b) In vivo, subcutaneous tumors were coelectroporated with siRNA‐Luc and pGL3‐Luc. After 48 h, tumors were excised and homogenized for bioluminescence measurement.

Figure 3

Inhibition of endogenous k‐ras expression after small interfering RNA (siRNA) treatment. (a) Two days after transfection, equal amounts of cell lysate were subjected to electrophoresis and western blot analysis. (b) Total RNA (1 µg) was subjected to semiquantitative reverse transcription–polymerase chain reaction with specific primers for either k‐ras or glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 4

Comparison of the antiproliferative effects induced by specific k‐ras oligodeoxynucleotide (ODN) antisense and k‐ras small interfering RNA (siRNA) on pancreatic tumor cell lines. The different tumor cell lines were untreated or treated with k‐ras antisense ODN, synthetic k‐ras siRNA or pSsik‐ras. Two days after transfection, cell proliferation assays were carried out by [3H]thymidine incorporation measurement (a) and the cytotoxic effects were evaluated using MTT cell viability tests (b). The assays were carried out in triplicate, and each result is representative of at least three independent experiments.

Figure 5

K‐ras downregulation inhibited cell viability and colony formation. (a) Panc1 tumor cells were treated over 48 h. Thereafter, cells were harvested and seeded in Petri dishes (60 mm) at different densities without any treatment. Approximately 10 days later, the plates were fixed with methanol and stained with crystal violet. (b) For each experiment, the colonies visualized by crystal violet staining were counted and the number was plotted in the graph as a percentage. Results are the means of three experiments ± SEM (***P < 0.001). (c) Ex vivo tumorigenesis assay. In vitro cell transfection was done as described in Materials and Methods. After 48 h, parental and small interfering RNA (siRNA)‐transfected Panc1 tumor cells were harvested and injected subcutaneously into nude mice, and the tumor volume was assessed twice weekly during 6 weeks. The left curve represents the tumor volume graph obtained after inoculation of untransfeted Panc1 (control) and transfected with pSsiGFP or pSsik‐ras^GAT. The right curve represents the results obtained with Capan1 tranfected in vitro with pSsiGFP or pSsik‐ras^GTT.

Figure 6

Silencing of K‐ras expression inhibited the growth of tumor xenografts in nude mice. Mice with subcutaneous BxPC3 (k‐ras^GGT) and Panc1 (k‐ras^GAT) tumors were randomized into groups of six animals and k‐ras antisense oligonucleoides and small interfeing RNA were delivered intratumorally by electropration two times. Individual mice were monitored for tumor growth over a period of 33 days. The volume for individual tumors was calculated and each curve represents the mean tumor volume calculated for each group (n = 6). Standard errors of the mean are represented by error bars.

Figure 7

Enhancement of gemcitabine antitumor effects by k‐ras small interfering RNA (siRNA). Panc1 pancreatic tumor cells (k‐ras^GAT) were implanted orthotopically. After 10 days, mice were assigned randomly into four groups. The siRNA k‐ras^GAT were delivered by intraperitoneal lipofection alone or in combination with gemcitabine (25 µg/kg). Gemcitabine in the single‐treatment protocol was administrated intraperitoneally at the dose 45 µg/kg. (a) Total tumor volume determination after 30 days showed a significant inhibition of tumor growth, particularly in pancreatic tumor‐bearing mice receiving combined treatment. (b) The general appearance of representative mice bearing orthotopic pancreatic tumor xenografts after 45 days. The control mouse (1) had a swelling abdomen and was larger than the mouse treated with pSsik‐ras^GAT (2), gemcitabine (3) or pSsik‐ras^GAT + gemcitabine (4). (c) Cumulative survival rate of athymic nude mice implanted orthotopically with Panc1 tumor cells. The survival curve was plotted according to the method of Kaplan–Meier (n = 10). Note the high prolongation of mouse survival by combined gemcitabine and k‐ras siRNA (pSsik‐ras^GAT) treatment compared with the control mice or with single agent‐treated mice.