Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion.

BACKGROUND: Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members. RESULTS: We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. CONCLUSIONS: Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.