Calphostin C

Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization.

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is generally used as a specific inhibitor of protein kinase C. It is known that 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, induces expression of mRNA for amphiregulin (AR), a member of EGF-related polypeptides, in mammalian epithelial cells. In this work, we determined the effect of calphostin C on AR mRNA expression in IEC-6 cells, a rat intestinal epithelial cell line, and unexpectedly found that this compound enhanced the TPA-induced expression of AR mRNA. Moreover, calphostin C alone induced expression of AR mRNA in a light-dependent manner, and this effect was abrogated by pretreatment with N-acetylcysteine. These results suggest that calphostin C can upregulate expression of AR mRNA via reactive oxygen species.

Perylenequinones that generate reactive oxygen species (ROS) when illuminated with visible light have been recommended as photodynamic chemotherapeutic agents. One of these is calphostin C (CalC), the action of the photo-activated derivative of which, CalCphiE, has been ascribed to its ability to selectively and irreversibly inhibit protein kinase Cs (PKCs). But recent results of experiments with neoplastic rat fibroblasts and human breast and uterine cervix cancer cells have revealed that the action of CalCphiE involves more than PKC inhibition. Besides suppressing PKC activity, CalCphiE rapidly causes endoplasmic reticulum (ER) stress in breast cancer cells and the selective complete oxidation and proteasomal destruction of the functionally essential nuclear envelope protein lamin B1, in human cervical carcinoma (HCC) cells and neoplastic rat fibroblasts. When these lamin B1-lacking cells are placed in the dark, cytoplasmic membrane-linked PKC activities suddenly rebound and apoptogenesis is initiated as indicated by the immediate release of cytochrome c from mitochondria and later on the activation of caspases. Hence, CalCphiE is a photodynamic cytocidal agent attacking multiple targets in cancer cells and it would be worth determining, even for their best applicative use, whether other perylenequinones also share the so far unexpectedly complex deadly properties of the CalCphiE.