Changxian Shen

FDG uptake in breast cancer: correlation with biological and clinical prognostic parameters

Abstract. The aim of this study was to evaluate the possible correlation between preoperative FDG-PET results in human breast cancer and the prognostic markers Ki-67, c-erb B2, p53, oestrogen/progesterone receptor status, axillary lymph node status, tumour size and tumour grading. Seventy-five female patients with breast cancer were included in this prospective study. Patient selection was independent of tumour size and the suspected clinical stage of disease. A high-resolution full-ring scanner (Siemens ECAT HR+) was used for PET imaging. The FDG uptake of breast tumours was calculated as the tumour to background ratio (TBR). In resected cancer tissue specimens, the proliferative fraction was evaluated by Ki-67 immunostaining. Additionally, immunostaining of the prognostic markers c-erb B2, p53, and progesterone and oestrogen receptors was performed. Haematoxylin and eosin-stained sections were used for tumour grading. Correlations between FDG uptake and prognostic markers were assumed to be significant at P<0.05 using the Mann-Whitney U test. In ductal breast cancer, mean TBR was 17.3 (median 7.7, range 1.6–122.7), while in lobular cancer it was 6.5 (median 3.7, range 1.4–22.7). Mean proliferative fraction (% Ki-67 positive tumour cells) was 15%±13.8% (median 10%, range 0%–60%). Twenty-three carcinomas showed <5% Ki-67 positive tumour cells. Statistical analysis indicated a positive correlation between FDG uptake and proliferative index in ductal breast cancer (P<0.0001, r=0.63). By contrast, there was no correlation between FDG uptake and c-erb B2 (P=0.79), p53 (P=0.92), tumour grading (P=0.09), oestrogen receptor status (P=0.41), progesterone receptor status (P=0.34), axillary lymph node status (P=0.90) and tumour size (P=0.3). It is concluded that FDG uptake is significantly higher in ductal breast cancer than in lobular cancer (P<0.05). FDG uptake correlates with proliferative activity assessed by Ki-67 immunostaining (P<0.05). A significant correlation with the other prognostic markers, however, could not be demonstrated.

Gene silencing by adenovirus-delivered siRNA

RNA interference is the process that double‐stranded RNA induces the homology‐dependent degradation of cognate mRNA mediated by 21–23 nucleotide short interfering RNA (siRNA). Here, we describe a simple virus vector for efficient delivery of siRNA into mammalian cells utilizing the well‐defined H1‐RNA promoter and conventional adenovirus. In this pilot study, p53 was targeted by this vector. Our results demonstrate efficient and specific knock‐down of p53 in breast cancer MCF‐7 and lung carcinoma A549 cells and indicate a prospective application of this siRNA expressing recombinant adenovirus system in functional genomics, cancer gene therapy and virus inhibition.

Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells

Survivin is expressed in most cancers but is undetectable in differentiated adult cells, and plays an important role both in the suppression of apoptosis and mitotic spindle checkpoint; thus it has attracted great interest as a potential drug target. In this study, we investigated the antigene and antiproliferative effects of triplex-forming oligodeoxynucleotides (TFO) targeting survivin in human lung carcinoma A549 cells. Survivin-specific TFOs form stable triplexes under physiological conditions as tested by electrophoretic mobility shift assays. Treatment of A549 cells with survivin-specific but not control TFOs at a concentration of 400 nM in the presence of uptake-enhancing liposome significantly reduced survivin protein level, inhibited cell proliferation, and induced cell apoptosis as demonstrated by immunoblot, cell number counting, and Annexin V-staining. Moreover, we found that the triplex-forming potential of TFOs measured in vitro does not necessarily correlate with the ability of TFOs to affect expression of a targeted gene in vivo. Our results indicate that targeting survivin is a promising alternative strategy for the development of novel anticancer therapeutics.

Targeting bcl-2 by triplex-forming oligonucleotide - A promising carrier for gene-radiotherapy

Triplex forming oligonucleotides (TFO) provide a promising tool for gene therapy. DNA damaging agents have been successfully coupled to TFOs and induce site-directed DNA damages. Here, we attempted to apply this antigen strategy using a TFO incorporated with a Conversion-electron-emitter, (99m)technetium, to target bcl-2 gene, the prototypical inhibitor of apoptosis. In the bcl-2 promoter region, we found two TFO binding sites which bind corresponding TFOs with very high specificity and affinity. Both partially and completely phosphorothioated TFOs form stable triplexes and significantly inhibit gene transcription in vitro. We also found that purine motif TFO with a thymidine opposite a thymidine interruption at the polypurine strand can form a stable triplex. In addition, (99m)technetium-conjugated TFOs were found to form a stable triplex and to inhibit bcl-2 gene transcription in vitro. Our results suggest a promising application of this triplex-forming oligonucleotide based Conversion-electron-emitter mediated gene radiotherapy in diseases related to bcl-2 overexpression.

Adenovirus-delivered siRNA.

RNA interference is the process that double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA mediated by 21- to 23-nt small interfering RNA (siRNA). Successful application of RNAi in functional genomics and proteomics, cancer gene therapy, and virus protection depends on the efficient delivery of siRNA into mammalian cells. The availability of high virus titer, infection of a broad spectrum of cell types, and independence on active cell division makes adenovirus the vector of choice for siRNA delivery. To this end, we developed a new adenovirus shuttle vector designated as pShuttle-H1 to host H1-RNA promoter and unique BglII and HindIII sites for insertion of oligos for expression of siRNA. In this chapter, we describe an adenovirus system that uses a commercially available adenovirus system and pShuttle-H1 to deliver siRNA-expressing cassette into cells to silence a specific gene in mammalian cells.

Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis.

AIM The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODNs). METHODS Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODNs using a polycationic liposome preparation. Specific hybridization of antisense ODNs was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis. RESULTS Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis. CONCLUSIONS Polycationic liposome-mediated transfection of bcl-2 antisense ODNs causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).