Induction of kai-1 expression in metastatic cancer cells by phorbol ester

H Akita, MDa, T Okamura,MD,a, A Iizuka,MD,b,Y Hashimoto,MD,b,K Tozawa,MD,b, K Kohri,MD,b

aUrology Division Meijo Hospital, Nagoya, Aichi Japan bDepartment of Urology, Nagoya City University Medical School,Nagoya, Aichi Japan

AIM: Although down-regulation of KAI-1 expression appears to be associated with the development of metastases or disease progression in several types of cancer, its molecular mechanism remains largely unknown. We determined whether the expression of KAI-1 mRNA can be induced in cancer cells by treatment with certain drugs. In the present study, we demonstrated that KAI-1 expression was induced by activating protein kinase C in various cancer cell lines. METHODS: 1)PMA induction of KAI-1 mRNA expression in various cell lines : mRNAs were isolated from Normal human fibroblasts (MJ-90) and several cancer cell lines. Northern blotting was performed using human KAI-1 cDNA as a probe. 2) PMA-mediated transcriptional up-regulation of the KAI-1 gene in U937 cells : Nuclei from U937 cells left untreated (Control) or treated with 20nM PMA were prepared and used for run-on transcription assays. 3) KAI-1 expression by activating PKC : DU145 cells were treated with the indicated compounds [0.1 % EtOH, 0.1 % DMSO, 20 nM 4aPDD, 20 nM PMA, and 20 nM calphostin C (Cal. C)]. After 24 hours of treatment, mRNAs were isolated and subjected to Northern blot analysis. RESULTS: KAI-1 expression was induced by PMA in various cell lines including metastatic prostate cancer cell lines, DU145, LNCap, and PC3 in which its expression was significantly down-regulated. KAI-1 expression was enhanced in a dose-dependent manner by PMA, and its induction was first detected at 6h after PMA treatment, reaching a plateau at 24h. A nuclear run-on assay revealed that the induction of KAI-1 expression by PMA is at least in part due to transcriptional activation. The inactive compound, 4aPDD, failed to induce KAI-1 expression and pretreatment with a specific inhibitor of protein kinase C (PKC), calphostin C, completely abrogated the induction of KAI-1 expression in DU145 cells. CONCLUSION: Our present study demonstrated that the KAI-1 gene can be induced through activation of the PKC pathway(s), even in metastatic cancer cells. Since KAI-1 protein expression is frequently downregulated at the transcriptional level in various types of human cancer cells and reintroduction of KAI-1 protein inhibited the appearance of the metastatic phenotype, it might be possible to synthesize analogs of PMA which inhibit metastasis by inducing KAI-1 expression but do not activate cancer cell growth.

KEY WORDS: prostate cancer, metastasis.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Metastasis.