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- Häggblad Sahlberg, Sara, et al.
(författare)
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The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines
- 2012
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 40:1, s. 176-184
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Tidskriftsartikel (refereegranskat)abstract
- The epidermal growth factor receptor (EGFR) is frequently overexpressed in colorectal cancer and is therefore an attractive target for treatment. (ZEGFR:1907)2 is a newly developed dimeric affibody molecule with high affinity to the extracellular part of EGFR. In this study, we evaluated the cytotoxic effects of (ZEGFR:1907)2 in combination with external radiation and the possible inhibitory effects in the EGFR signalling pathways in the colon cancer cell lines HT-29 and HCT116. The effects were compared with an EGFR antibody (cetuximab) and the tyrosine kinase inhibitors (erlotinib and sunitinib). These cell lines are genotypically different with respect to e.g. KRAS and BRAF mutational status, recently shown to be of clinical significance for therapeutic effects. Both cell lines express approximately 100,000-150,000 EGFRs per cell but differ in the radiation response (HCT116, SF2=0.28 and HT-29, SF2=0.70). Exposure to (ZEGFR:1907)2 produced a small, but significant, reduction in survival in HCT116 but did not affect HT-29 cells. Similar results were obtained after exposure to EGF and the EGFR antibody cetuximab. The EGFR tyrosine kinase targeting inhibitor erlotinib and the multi-tyrosine kinase inhibitor sunitinib reduced survival in both cell lines. However, none of the drugs had any significant radiosensitizing effects in combination with radiation. Akt and Erk are central proteins in the EGFR downstream signalling and in the cellular response to ionizing radiation. The activation of Akt (Ser 473) and Erk (Thr202/Tyr204) by radiation was both dose- and time-dependent. However the activation of EGFR was not clearly affected by radiation. Neither (ZEGFR:1907)2 nor any of the other drugs were able to completely inactivate Akt or Erk. On the contrary, erlotinib stimulated Akt phosphorylation in both cell lines and in HCT116 cells Erk was activated. Overall the results illustrate the complexity in response to radiation and drugs in cells with differential phenotypic status.
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| 2. |
- Abel, Frida, 1974, et al.
(författare)
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Mutations in the N-terminal domain of DFF45 in a primary germ cell tumor and in neuroblastoma tumors.
- 2004
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Ingår i: International journal of oncology. - 1019-6439 .- 1791-2423. ; 25:5, s. 1297-302
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Tidskriftsartikel (refereegranskat)abstract
- DFF45 has essential functions in the final stage of apoptosis by acting both as a folding chaperone and a DNase inhibitor of DFF40. The gene encoding DFF45 (DFFA) maps to the consensus deleted region in primary neuroblastoma (NB; 1p36.2-3) and within the homozygously deleted region in an NB cell line (1p36.2). DFF45 is therefore an attractive candidate NB tumor suppressor. In a previous study we found a rare allele variant, causing a non-polar to a polar amino acid exchange (Ile69Thr) in a preserved hydrophobic patch of DFF45, and we also found DFFA to be preferentially expressed in favorable NB tumors. We have extended the previous study and performed mutation analyses in another 56 NB tumors (100 in total) as well as a set of other tumors for coding mutations in DFFA. We have also performed studies of the DFFA expression in tumors using real-time PCR. We found a missense mutation (Ile15Met) in the remaining allele of a teratoma with heterozygous deletion of 1p, and a three base-pair deletion in an NB of unknown stage causing a deletion of amino acid 37 in DFF45. The one-base substitution detected in the teratoma was not present in the patients constitutional DNA, i.e. it is a true mutation present in the tumor DNA only. In conclusion, three different coding alterations have been found in the region encoding the N-terminal regulatory domain of DFF45, responsible for binding and achieving its chaperone and inhibitor functions on other proteins. Moreover, by real-time RT-PCR expression study, we found the mRNA level of DFFA to be significantly (p=0.038) reduced by a factor of 1.7 times in NB tumors of unfavorable outcome.
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| 4. |
- Ahnström, Marie, et al.
(författare)
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Altered expression of cyclin E and the retinoblastoma protein influences the effect of adjuvant therapy in breast cancer
- 2009
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 34:2, s. 441-448
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Tidskriftsartikel (refereegranskat)abstract
- Cyclin E and the retinoblastoma protein (Rb) are both important regulators of the G(1) phase in the cell cycle. Overexpression of cyclin E and lost expression of Rb has previously been observed in breast tumours at frequencies of 10-50% and 20-30%, respectively. We explored the prognostic role of cyclin E and Rb in breast cancer patients randomised for tamoxifen (TAM), CMF (cyclophosphamide, metotrexate, 5-fluorouracil) chemotherapy and radiotherapy (RT) and how their expression affects the patients response to treatment. Protein expression was assessed with immunohistochemistry. We found overexpression of cyclin E in 32.1% (71/221) of the tumours and loss of Rb expression in 25.0% (59/236). Increased expression of cyclin E correlated to dysfunctional p53 (P=0.003) while loss of Rb correlated to normal p53 status (P=0.001). Our results suggest that patients with high cyclin E tumours have less benefit from tamoxifen (ER+, TAM vs. no TAM; RR=0.97; 95% CI, 0.36-2.60) than patients whose tumours show low expression (ER+, TAM vs. no TAM; RR =0.41; 95% CI, 0.24-0.72). Cyclin E also tended to predict the benefit from radiotherapy with a local recurrence rate of 0.31 (RT vs. CMF; 95% CI, 0.12-0.93) for patients with low expression and 0.68 (RT vs. CMF; 95% CI, 0.2-2.32) for patients with high expression of cyclin E. When the p53 status was taken in consideration the results showed that patients with both normal p53 and normal Rb expression had considerably lower locoregional recurrence rate when treated with radiotherapy instead of CMF (RR=0.17; 95% CI, 0.052-0.58) as compared to patients with either altered Rb or p53 or both (RR=0.70; 95% CI, 0.28-1.73).
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| 6. |
- Akhtar, Monira, et al.
(författare)
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Cell type and context-specific function of PLAG1 for IGF2 P3 promoter activity
- 2012
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 41:6, s. 1959-1966
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Tidskriftsartikel (refereegranskat)abstract
- The fetal transcription factor PLAG1 is found to be overexpressed in cancers, and has been suggested to bind the insulin like growth factor 2 (IGF2) P3 promoter, and to activate the IGF2 gene. The expression of IGF2 has partly been linked to loss of CTCF-dependent chromatin insulator function at the H19 imprinting control region (ICR). We investigated the role of PLAG1 for IGF2 regulation in Hep3B and JEG-3 cell lines. Chromatin immunoprecipitation revealed cell type-specific binding of PLAG1 to the IGF2 P3 promoter, which was substantially insensitive to recombinant PLAG1 overexpression in the endogenous context. We hypothesized that the H19 chromatin insulator may be involved in the cell type-specific PLAG1 response. By using a GFP reporter gene/insulator assay plasmid construct with and without the H19 ICR and/or an SV40 enhancer, we confirm that the effect of the insulator is specifically associated with the activity of the IGF2 P3 promoter in the GFP reporter system, and furthermore, that the reporter insulator is functional in JEG-3 but not in Hep3B cells. FACS analysis was used to assess the function of PLAG1 in low endogenously expressing, but Zn-inducible stable PLAG1 expressing JEG-3 cell clones. Considerable increase in IGF2 expression upon PLAG1 induction with a partial insulator overriding activity was found using the reporter constructs. This is in contrast to the effect of the endogenous IGF2 gene which was insensitive to PLAG1 expression in JEG-3, while modestly induced the already highly expressed IGF2 gene in Hep3B cells. We suggest that the PLAG1 binding to the IGF2 P3 promoter and IGF2 expression is cell type-specific, and that the PLAG1 transcription factor acts as a transcriptional facilitator that partially overrides the insulation by the H19 ICR.
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| 10. |
- Altai, Mohamed, et al.
(författare)
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Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin
- 2016
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 49:3, s. 1185-1194
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Tidskriftsartikel (refereegranskat)abstract
- Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide In-111. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)(3) purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, In-111-(HE)(3)-Z(HER2)-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.
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