Anticancer activity of RecQL1 helicase siRNA in mouse xenograft models

Abstract

Small interfering RNAs (siRNAs) are expected to have a medical application in human therapy as drugs with a high specificity for their molecular target mRNAs. RecQL1 DNA helicase in the human RecQ helicase family participates in DNA repair and recombination pathways in the cell cycle of replication. Silencing the RecQL1 expression by RecQL1-siRNA induces mitotic death in vitro specifically in growing cancer cells. By contrast, the same RecQL1 silencing does not affect the growth of normal cells, emphasizing that RecQL1 helicase is an ideal molecular target for cancer therapy. In this study, we show that local and systemic administration of RecQL1-siRNA mixed with polyethyleneimine polymer or cationic liposomes prevented cancer cell proliferation in vivo in mouse models of cancer without noticeable adverse effects. The results indicate that RecQL1-siRNA in a complex with a cationic polymer is a very promising anticancer drug candidate, and that in particular, RecQL1-siRNA formulated with a cationic liposome has an enormous potential to be used by intravenous injection for therapy specific for liver cancers, including metastasized cancers from the colon and pancreas.

Figure 1

Expression of RecQL1 helicase in the liver cancer tissue arrays classified from grade I to III based on progression of cancer. A total of 60 human liver cancer specimens classified from grade I to III (provided by Cybrdi, Frederick, MA, USA) were stained immunohistochemically by an antibody specific to RecQL1.^{( 17 )} The level of staining was compared between the grades and with normal liver tissues (n = 3) that were not stained under the same conditions. (a) Progression of cancers classified by supplier of the liver cancer array; (b₁) normal liver tissue –; (b₂) high degree of staining +; (b₃) higher degree of staining ++; (b₄) highest degree of staining +++; (c) immunohistochemical staining of RecQL1 classified in this study.

Figure 2

Anticancer effect of RecQL1‐siRNA/PEI complex on subcutaneously inoculated lung cancer cells in mouse model by local and systemic administration. (a) A549 human lung cancer cells were inoculated under the dorsal skin of mice as described in the ‘Materials and Methods’, and cancer nodules formed after 8 days were injected subcutaneously with RecQL1‐siRNA (25 µg) mixed with polyethylenimine carrier (5 µg) in 50 µL saline. NS‐siRNA was similarly injected as a negative control. Injections were made on days 8, 11, 14, 17, 20, 23, 27, and 32 (arrow heads). Six mice formed a group and their average size of cancer nodule was plotted. The blue diamonds show NS‐siRNA/PEI complex and the pink squares show RecQL1‐siRNA/PEI complex. *indicates the statistical significance at P < 0.05. (b) Comparison of the size of A549 lung cancer nodules formed at 50 days after cancer cell inoculation. NS‐siRNA/PEI complex‐treated cancer nodules (upper); RecQL1‐siRNA/PEI complex‐treated cancer nodules (lower). (c) Detection of TdT‐mediated dUTP nick end‐labeling (TUNEL) staining‐positive cells in cancer nodules. Sliced sections of A549 cancer nodules were stained with hematoxylin–eosin. Cells undergoing apoptotic‐like death were detected by TUNEL staining. (c₁) NS‐siRNA/PEI complex‐treated cancer nodule; (c₂) RecQL1‐siRNA/PEI complex‐treated cancer nodule. (d) Effect of RecQL1‐siRNA/PEI complex on subcutaneously inoculated lung cancer A549 cells in a mouse model by systemic administration. RecQL1‐siRNA/PEI complex (containing 50 µg RecQL1‐siRNA) was injected intravenously into the tail vein. Injections were made on days 8, 11, 14, 17, 20, 23, 27, and 32 (arrow heads). Six mice formed one group, and average cancer nodule volumes are shown. Open triangles, solid squares, and open circles indicate mice treated with saline, NS‐siRNA/PEI, and RecQL1‐siRNA/PEI complex, respectively. (e) Retention of the RecQL1‐siRNA/PEI complex in the circulation blood of mice. RecQL1‐siRNA fluorescently labeled with tetramethylrhodamine (TAMRA) at the 5′‐terminus of the sense strand was mixed with PEI, and the retention time in the circulating blood was investigated according to the method described by Sato et al.^{( 31 )} The labeled RecQL1‐siRNA/PEI complex was injected into the tail vein and was extracted from the eye vein at 1, 5, and 10 min after the injection. Parts of the extracted blood samples were extracted with phenol‐chloroform and were analyzed by using 10% polyacrylamide gel electrophoresis. As a control, fluorescent RecQL1‐siRNA without PEI was also similarly analyzed. RecQL1‐siRNA mixed with PEI (PEI). RecQL1‐siRNA without PEI (naked).

Figure 3

Anticancer effect of the RecQL1‐siRNA/LIC‐101 complex on liver cancer in a mouse model by local administration. (a) Time course for measuring the inhibitory effects of RecQL1‐siRNA/LIC‐101 complex on the growth of Hep3B cancer nodule. Hep3B cells were inoculated under the dorsal skin of mice, and the RecQL1‐siRNA/LIC‐101 complex was injected subcutaneously as described in the Figure 1 legend. The injections were made on days 14, 16, 18, 21, 23, 25, 27, 32, and 36 (shown by arrow heads). Seven mice formed one group and average sizes of cancers are shown. Open squares, closed triangles, and closed circles indicate mice treated with 10% maltose, NS‐siRNA/LIC‐101, and RecQL1‐siRNA/LIC‐101 complex, respectively. *indicates the statistic significance at P < 0.05. (b₁) Mice treated with 10% maltose (w/v) solution (42 days after inoculation), (b₂) Mice treated with NS‐siRNA/LIC‐101 complex (42 days); (b₃) Mice treated with RecQL1‐siRNA/LIC‐101 complex (42 days). (c) Tissue stained by hematoxylin–eosin. (c₁) NS‐siRNA/LIC‐101 complex‐treated cancer nodule, (c₂) RecQL1‐siRNA/LIC‐101 complex‐treated cancer nodule. (d) Immunohistochemical staining with RecQL1‐specific antibody. (d₁): NS‐siRNA/LIC‐101 complex‐treated cancer nodule, (d₂) RecQL1‐siRNA/LIC‐101 complex‐treated cancer nodule.

Figure 4

Inhibiton of growth of AsPC‐1 pancreatic cancer cells by systemic administration of the RecQL1‐siRNA/LIC‐101 complex in a mouse model of metastasized liver cancer. (a) Inhibition of AsPC‐1 cell proliferation in vitro by RecQL1 silencing. AsPC‐1 cells were transfected in vitro by RecQL1‐siRNAs with Lipofectamine 2000, and the inhibitory effect on proliferation was investigated. The viability of cells was measured by colorimetric assay as described in the ‘Materials and Methods’. Open triangles show NS‐siRNA–treated cells, and closed circles show RecQL1‐siRNA‐treated cells. The histograms show levels of RecQL1 mRNA in the RecQL1‐silenced cells that are represented as a proportion (%) of NS‐treated cells. (b) AsPC‐1 cells with 40 nM RecQL1‐siRNA in the presence of Lipofectamine 2000 were analyzed by using flow cytometry after 72 h of culture. NS, Non‐silencing siRNA‐treated cells; RecQL1, RecQL1‐siRNA‐treated cells. (c) Photographs of mouse livers treated with the NS‐siRNA/LIC‐101 complex and RecQL1‐siRNA/LIC‐101 complex at 29 days after intrasplenic inoculation with AsPC‐1 cells. (c₁) 10% maltose, (c₂) NS‐siRNA/LIC‐101 complex, (c₃) RecQL1‐siRNA/LIC‐101 complex‐treated liver. (d) Anticancer effect of the RecQL1‐siRNA/LIC‐101 complex evaluated by liver weight change in mouse liver metastasis model. AsPC‐1 cells were inoculated into mouse liver intrasplenically on day 0. The RecQL1‐siRNA/LIC‐101 complex was administrated as described in ‘Materials and Methods’. Liver weights were measured on day 29. *Mean SE (n = 10) is the significant difference from the maltose by Dunnett's test; P < 0.05. NS; NS‐siRNA/LIC‐101 complex, QL1; RecQL1‐siRNA/LIC‐101 complex‐treated, sham; sham‐operation. (e,f) Cancer markers CYFRA 21‐1 and CEA indicating the anticancer effect of RecQL1‐siRNA/LIC‐101 complex intravenously injected into mice having pancreatic cancer cells metastasized to liver. *Mean SE (n = 10) is the significant difference from maltose by Dunnett's test; P = 0.0016 (CYFRA 21‐1) and 0.0029 (CEA). (g) Expression of RecQL1 mRNA in human AsPC‐1 cancer cells metastasized in liver determined on day 29 by reverse transcription–polymerase chain reaction (RT‐PCR). This PCR analysis strictly precludes the spill‐over from mouse RecQL1 mRNA. (h) Expression of RecQL1 protein in human AsPC‐1 cancer cells metastasized in the liver determined on day 29 by Western blotting. The AsPC‐1 cells metastasized in the mouse liver were homogenized, and the portion (containing 50 µg protein) was analyzed for human RecQL1 protein expression by using antibodies specific for human RecQL1 and actin. The antibody to human actin, though weakly, cross reacted with mouse actin.

Figure 5

Inhibition of LS174T colon cancer cell growth by intravenous injection of the RecQL1‐siRNA/LIC‐101 complex in a mouse model of metastatic liver cancer. (a) Livers treated with the NS‐siRNA/LIC‐101 complex and RecQL1‐siRNA/LIC‐101 complex at 37 days after intrasplenic inoculation with LS174T. (a₁) sham operation; (a₂) 10% maltose; (a₃) NS‐siRNA/LIC‐101 complex; (a₄) RecQL1‐siRNA/LIC‐101 complex. (b) LS174T was intrasplenically inoculated into nude mice on day 0. The RecQL1‐siRNA/LIC‐101 complex was administrated as described in ‘Materials and Methods’. Liver weights were measured on day 37. *Mean SD (n = 7) is the significant difference from the maltose by Dunnett's test; P < 0.05. (c) Cancer marker CYFRA 21‐1 indicating the anticancer effect of RecQL1‐siRNA/LIC‐101 complex intravenously injected into mice having liver metastasis cancer. *Mean SE (n = 7) is the significant difference from the maltose by Dunnett's test; P = 0.008.

Figure 6

Distribution of siRNA/LIC‐101 complex and toxicological studies. (a) Kinetics of retention of siRNA/LIC‐101 complex in circulating blood: after the intravenous injection, RecQL1‐siRNA was extracted from the blood and was quantitated by using the method described in the ‘Materials and Methods’. (b) Distribution of siRNA in organs: After the intravenous injection, RecQL1‐siRNA was extracted from various organs 15 min after administration and was quantitated by using the method described in the ‘Materials and Methods’. (c) Distribution of siRNA in organs: Alexa‐546 labeled‐siRNA/LIC‐101 complex (1 mg/kg) was administrated into BALB/c mice by intravenous injection into the tail vein. Distribution of siRNA in organs was visualized by the fluorescence after 24 h. (d) Distribution of intravenously injected FAM‐labeled‐siRNA/LIC‐101 complex in the liver after 24 h. (d₁) non‐cancer cell region; (d₂) cancer cell region. Photographic pictures of the stained sliced liver are shown. Arrow heads show the FAM‐labeled‐siRNA, and blue dots show the nuclei of the non‐cancer cells selectively stained by Hoechst33342. (e) Toxicological studies with liver and kidney: 2 or 10 mg/kg of RecQL1‐siRNA/LIC‐101 complex was given to BALB/c mice every other day for 4 weeks. The serum was investigated for liver and kidney function markers. Data represent the mean ± SD (n = 5). (f) Pathological studies on mice injected with the RecQL1‐siRNA/LIC‐101 complex (intravenous administration of 10 mg/kg every other day for 4 weeks). Liver slices were stained by hematoxylin–eosin. (f₁) 10% maltose, (f₂) RecQL1‐siRNA/LIC‐101 complex. (g) Absence of interferon‐alpha (IFN‐α) induction by RecQL1‐siRNA/LIC‐101 liposome. BALB/c mice were administrated with RecQL1‐siRNA (50 µg) together with LIC‐101 liposome in 10% maltose by intravenous injection into the tail vein. The positive control group was injected with poly (I:C). Serum was collected 3 h after injection; the IFN‐α level was determined by ELISA.