Inhibition of protein glycosylation reverses the MDR phenotype of cancer cell lines.
BACKGROUND
Multidrug resistance proteins are one of the most important factors that cause chemotherapy resistance, which in turn reduces therapeutic efficacy and survival for cancer patients. Tunicamycin is one of the most well-known inhibitors of N-glycosylation and is considered a powerful adjunct that can increase the effectiveness of many drugs. Tunicamycin blocks the first step of P-gp (glycoprotein P) and BCRP (breast cancer resistance protein) N-glycosylation, which is a very important modification for the activity and cellular localisation of these proteins.


METHODS
The effects of tunicamycin on ovarian and colorectal cancer cells were examined in multiple cell lines. The primary ovarian cancer cell line W1 and the established ovarian cancer cell line A2780 were compared against their drug-resistant derivatives W1TR/W1PR (TR: topotecan resistant; PR: paclitaxel resistant) and A2780T1 (topotecan resistant), respectively. We also compared the colorectal cancer cell line LoVo against its doxorubicin-resistant derivative LoVo/Dx. Cell viability was determined by the MTT assay. The glycopeptides were subjected to deglycosylation using the endoglycosidase PNGase F. A2780T1, LoVo/Dx and W1PR cells were treated with the protein degradation inhibitors MG132 and BMA. Protein expression was detected by western blot and immunocytochemistry.


RESULTS
In this study, we showed via the MTT assay that tunicamycin significantly decreased the viability of cancer cell lines that were co-treated with a chemotherapeutic drug. Western blot analysis showed that, in LoVo/Dx and W1PR cells, tunicamycin treatment resulted in the expression of a 70kDa P-gp protein instead of the mature 170kDa P-gp. Treatment with MG132 or BMA fully suppressed the effect of tunicamycin in the case of W1PR cells only. In tunicamycin-treated W1TR cells, the size of the BCRP protein did not differ from that of its native unglycosylated form. In tunicamycin-treated A2780T1 cells, BCRP expression was completely inhibited, but pre-treatment with MG132 or BMA suppressed the effect of tunicamycin. Immunocytochemistry analysis indicated that tunicamycin only affected the translocation of P-gp but not that of BCRP. After treatment, we observed higher P-gp expression in the cytoplasm than at the cell membrane.


CONCLUSIONS
Our results indicated that tunicamycin may enhance the effect of chemotherapy by interfering with the localisation and function of transporter proteins that are responsible for multidrug resistance.
Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland. Electronic address: k_wojtowicz@onet.pl.
Karolina
K
Wojtowicz
Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland. Electronic address: rjanuchowski@wp.pl.
Radosław
R
Januchowski
Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland. Electronic address: mnowicki@ump.edu.pl.
Michał
M
Nowicki
Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; Department of Histology and Embryology, Wroclaw Medical University, 50-368 Wroclaw, Poland. Electronic address: mazab@ump.edu.pl.
Maciej
M
Zabel
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology; D010641:Phenotype; D019772:Topotecan / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology*
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D014415:Tunicamycin; D019772:Topotecan; D004317:Doxorubicin; D017239:Paclitaxel
BCRP; Chemotherapy; Glycoprotein P; Multidrug resistance; Tunicamycin