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Secondary stimulation from Bacillus Calmette-Guérin induced macrophages upregulatesNOS2 protein in bladder cancer cells
Karolinska Institutet.
(Karolinska Institutet)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Karolinska Institutet.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Treatment with Bacillus Calmette-Guérin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG-instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG-mediated effect. Using immunohistochemistry we have previously shown nitric oxide synthase 2 (NOS2/iNOS) protein expression in both immune cells and in urothelial cells in bladder cancer patient biopsies. In this study we analysed the influence of macrophage- (RAW 264.7) secreted factors on NO production by stimulating urothelial carcinoma cells (MBT2) with supernatant from BCG-treated macrophages as well as supernatant from untreated macrophages. Using real-time PCR, western blot and chemiluminescence, we found no effect of BCG when added straight to the culture medium of urothelial carcinoma cells. However, when 40% of the culture medium of the bladder cancer cells was substituted with supernatant from BCGstimulated macrophages, we found increased NOS2 mRNA and protein expression as well as increased levels of NO. In addition we found increased cell death only in bladder cancer cells stimulated with supernatant from BCG-treated macrophages, as visualized by cell cycle analysis and PARP cleavage. These results suggest that simultaneous targeting of the microenvironment and subsequent stimulation of adaptive responses can improve conventional BCG-therapy.

Keyword [en]
nitric oxide synthase type II, BCG vaccine, urinary bladder neoplasms, nitric oxide
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-79014OAI: oai:DiVA.org:su-79014DiVA: diva2:546577
Available from: 2012-08-24 Created: 2012-08-23 Last updated: 2012-08-27Bibliographically approved
In thesis
1. Studies of bioactive lipids in cancer
Open this publication in new window or tab >>Studies of bioactive lipids in cancer
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lipids are a broad class of molecules that, besides being a major form of energy storage and components of cell membranes, act as bioactive signalling molecules. N-acyl taurines are structurally related to endocannabinoids that are known to exert antiproliferative actions in a variety of cancer cells. We have evaluated the cytotoxicity of N-oleoyl taurine and N-arachidonoyl taurine and found N-acyl taurines to reduce proliferation of prostate cancer cells.

The sphingolipids ceramide and sphingosine act as tumour suppressors. We found selenite treatment to cause reduced viability, induction of neutral sphingomyelinase activity and accumulation of ceramide in the liver cancer cells. Inhibition of sphingosine kinase 1 (SK1) sensitized the cells to selenite treatment with regards to ceramide accumulation, arrest of cells in the G1/S phases of the cell cycle and formation of reactive oxygen species. Whereas combined selenite treatment and SK1 inhibition synergistically reduced the number of viable cells, the non-tumorigenic hepatocyte cell line, remained unaffected.

Furthermore, we studied the involvement of sphingolipid metabolism in bladder cancer cells treated with Bacillus Calmette-Guérin (BCG), and found BCG to induce formation of nitric oxide and upregulation of nitric oxide synthase 2 as well as SK1 protein levels. Additionally, pharmacological inhibition of SK1 enhanced the viability reduction, ceramide accumulation and induction of apoptosis observed following BCG treatment.

In conclusion, our findings have shown that N-acyl taurines exert antiproliferative effects on prostate cancer cells. Furthermore, sphingolipids were shown to be involved in cytotoxic treatment with selenite and BCG in liver cancer and bladder cancer cells respectively, and inhibition of SK1 increased the cytotoxicity. Our findings raise the possibility that modulation of ceramide-metabolizing enzymes could be used to enhance the effects of selenite and BCG in these cancer cell types.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 67 p.
Keyword
Lipids, Sphingolipids, N-acyl amino acids, cancer
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-79024 (URN)978-91-7447-548-7 (ISBN)
Public defence
2012-09-26, lecture room G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2012-09-04 Created: 2012-08-23 Last updated: 2012-08-27Bibliographically approved

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