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Molecular Studies of Irradiation and SN-38 on Colorectal Cancer
Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments.

The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV).

In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2008. , 60 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1070
Keyword [en]
Colorectal cancer (CRC), molecular changes, SN-38
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:liu:diva-14789ISBN: 978-91-7393-838-9 (print)OAI: oai:DiVA.org:liu-14789DiVA: diva2:25268
Public defence
2008-09-29, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2009-08-22Bibliographically approved
List of papers
1. Expression of PRL proteins at invasive margin of rectal cancers in relation to preoperative radiotherapy
Open this publication in new window or tab >>Expression of PRL proteins at invasive margin of rectal cancers in relation to preoperative radiotherapy
2006 (English)In: International Journal of Radiation Oncology, Biology, Physics, ISSN 0360-3016, E-ISSN 1879-355X, Vol. 65, no 2, 452-458 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: PRL-3 (phosphatase of regenerating liver) is involved in metastasis of colorectal cancer; however, its therapeutic implication in cancer patients has not been studied. We investigated the relationships of PRL expression to radiotherapy (RT) in rectal cancer patients.

Methods and Materials: Phosphatase of regenerating liver expression was immunohistochemically examined in distant (n = 36) and adjacent (n = 82) normal mucosa, primary tumor (n = 125), biopsy specimens (n = 96), and lymph node metastasis (n = 30) from rectal cancer patients participating in a clinical trial of preoperative RT.

Results: Phosphatase of regenerating liver expression was increased from the distant to adjacent mucosa and to the primary tumor (p < 0.05). PRL was highly expressed at the invasive margin in 28% of the primary tumors and 26% of the metastases. In the RT group, strong PRL expression at the invasive margin was related to distant recurrence (p = 0.006) and poor survival (p = 0.01), but not in the non-RT group. The survival significance remained even after adjusting for Dukes’ stage and differentiation (p = 0.02). Additional multivariate analyses showed that the correlation with prognostic significance of PRL differed between the RT and non-RT groups (p = 0.01).

Conclusion: Phosphatase of regenerating liver expression (rather than PRL-3 alone) at the invasive margin predicted resistance to RT and unfavorable survival in rectal cancer patients with preoperative RT.

Keyword
Phosphatase of regenerating liver; PRL; Invasive margin; Radiotherapy; Prognosis; Rectal cancer
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14784 (URN)10.1016/j.ijrobp.2005.12.043 (DOI)16626893 (PubMedID)
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13Bibliographically approved
2. Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells with mutant p53
Open this publication in new window or tab >>Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells with mutant p53
2009 (English)In: Journal of Cancer Research and Clinical Oncology, ISSN 0171-5216, E-ISSN 1432-1335, Vol. 135, no 11, 1583-1592 p.Article in journal (Refereed) Published
Abstract [en]

We previously found that p53, p73, survivin and PRL were implicated in the outcome of radiotherapy in rectal cancer patients. In the present study, we tried to understand mechanisms of colon cancer cell line response to radiation based on protein expression related to proliferation and apoptosis. KM12C, KM12SM and KM12L4a, cell lines with one origin, were radiated with 0, 10 or 15 Gy γ-radiation. Radiosensitivity was determined with cell cycle and apoptosis analysis, and protein expression of TAp73, ΔNp73, mutated p53, survivin and PRL-3 was determined by Western blot. KM12C showed transient G2-arrest, low apoptosis and up-regulation of resistance factors such as PRL-3. In KM12C expression of ΔNp73 increased after 10Gy, but not after 15Gy. KM12SM had permanent G2-arrest, low apoptosis and showed up-regulation of the anti-apoptotic survivin and down-regulation of the pro-apoptotic TAp73 and the radioresistance factor PRL-3 was down-regulated. KM12L4a, the most radiosensitive cell line, showed up-regulation of TAp73 and down-regulation/no up-regulation of resistance factors such as ΔNp73, survivin and PRL-3 after radiation. In conclusion, the KM12C cell line was more radioresistant than KM12L4a regarding apoptosis and certain apoptotic proteins. The radiosensitivity of KM12L4a might partly depend on the lack of up-regulation of proteins negative for the outcome of radiotherapy.

 

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14786 (URN)10.1007/s00432-009-0606-4 (DOI)
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13Bibliographically approved
3. Anticancer effect of SN-38 on colon cancer cell lines with different metastatic potential
Open this publication in new window or tab >>Anticancer effect of SN-38 on colon cancer cell lines with different metastatic potential
Show others...
2008 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 19, no 6, 1493-1498 p.Article in journal (Refereed) Published
Abstract [en]

SN-38 is an active metabolite of the topoisomerase I inhibitor irinotecan. The mechanism behind its antitumor effect in colorectal cancer is not fully understood. In this study, we examined the response of colon cancer cell lines with different metastatic potential to SN-38. The parental human colon cancer cell line KM12C and its two highly metastatic derivatives KM12SM and KM12L4a were cultivated in 5% CO2 at 37°C for 24 h and then exposed to SN-38 (2.5 µg/ml) at 37°C for 4, 24 and 48 h, respectively. The cell cycle was measured by flow cytometry, apoptotic activity was determined by flow cytometry and immunocytochemistry and the expression of topoisomerase I, Bax and survivin proteins were examined by Western blot. The exposure of the cells to SN-38 induced S-phase and G2 arrest (P<0.0001) and the KM12L4a cells had the highest response in a time-dependent manner (P<0.0001). The rates of apoptosis in the KM12SM (P=0.001) and KM12L4a cell lines (P=0.01) were increased time-dependently, though there was no such change in the KM12C cells. The expression of topoisomerase I protein was decreased in each cell line tested and the expression of Bax protein was increased, especially in KM12L4a. In conclusion, the effect of SN-38 on the colon cancer cell lines was mediated via conducting S-phase and G2 arrest and apoptosis. This effect was found in the cell lines with higher metastatic potentials, indicating that SN-38 can be used to treat advanced colon cancers.

Keyword
Colon cancer cell lines, SN-38, apoptosis
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14787 (URN)10.3892/or.19.6.1493 (DOI)
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13
4. Gene expression profile of colon cancer cell lines treated with SN-38
Open this publication in new window or tab >>Gene expression profile of colon cancer cell lines treated with SN-38
Show others...
2010 (English)In: Chemotherapy, ISSN 0009-3157, E-ISSN 1421-9794, Vol. 56, no 1, 17-25 p.Article in journal (Refereed) Published
Abstract [en]

Aim: Colorectal cancer is the third most common form of cancer in the industrialcountries. Due to advances regarding the treatments, primarily development ofimproved surgical methods, and the ability to make the earlier diagnosis, the mortalityhas remained constant during the past decades even though the incidence in fact hasincreased. To improve chemotherapy and enable personalised treatment, the need ofbiomarkers is of great significance. In this study we evaluated the gene expressionprofiles of the colon cancer cell lines treated with SN-38, the active metabolite oftopoisomerase-1 inhibitor irinotecan which leads to cell cycle arrest and apoptosis.Material and Methods: The study included three colon cancer cell lines KM12C,KM12SM and KM12l4a. The three cell lines were treated with SN-38, and sampleswere obtained after 24 and 48 hour treatments. The gene expression analyses wereperformed using oligonucleotide microarrays comprising of ~27,000 spots where theuntreated controls were compared to the SN-38 treated samples. Results: Unsupervisedclustering clearly distinguished the treated cell lines from the untreated. Supervisedanalysis identified 3974 significant genes (p=0.05) differentiating the treated samplesfrom the untreated, majority of which were downregulated after treatment. The toprankeddownregulated genes in the treated cell lines included those related to receptorand kinase activity, signal transduction, apoptosis, RNA processing, protein metabolismand transport, cell cycle and transcription. A smaller number of genes were upregulatedin the cell lines after treatment and included genes involved in apoptosis, transcription,development and differentiation. Conclusions: These results demonstrate that theexpression of the genes involved in cell proliferation and apoptosis as well as RNA,DNA and protein metabolism were affected by SN-38. The impact of certain genes oncolorectal cancer development needs to be further evaluated, however these resultscould serve as a basis for further studies in order to find targets for irinotecan treatment.

Place, publisher, year, edition, pages
S. Karger AG, 2010
Keyword
Colorectal, oncology, cancer
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14788 (URN)10.1159/000287353 (DOI)000276593500003 ()
Note
Original Publication: Åsa Wallin, P. Francis, N. Nilbert, Joar Svanvik and Xiao-Feng Sun, Gene expression profile of colon cancer cell lines treated with SN-38, 2010, Chemotherapy, (56), 1, 17-25. http://dx.doi.org/10.1159/000287353 Copyright: S. Karger AG http://www.karger.com/ Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13

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