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Protein kinase C in adriamycin action and resistance in mouse sarcoma 180 cells
J Posada1, P Vichi, T R Tritton
1Department of Pharmacology, University of Vermont School of Medicine, Burlington 05405.
Abstract:
Adriamycin has a wide variety of biological actions on susceptible cells, several of which may be integrally involved in cytotoxicity. In this paper, we present evidence that one of the alterations in cell function that occurs in the presence of Adriamycin is an elevation in the production of diacylglycerol. The effect is rapid, reaches a peak within 10 min of exposure of Sarcoma 180 cells to Adriamycin, and can thus be classified among the earliest alterations that occur in cells damaged by Adriamycin. Concomitant with the rise in diacylglycerol is an increase in cytosolic protein kinase C activity. Although Adriamycin does not appear to modulate the activity of this enzyme by direct binding, drug-exposed Sarcoma 180 cells have a 56% increase in intrinsic cytosolic protein kinase C (PKC) activity, with no change in the activity of the membrane form. Experiments with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggest that the PKC effect is linked to Adriamycin action, since activation of the enzyme by short 12-O-tetradecanoylphorbol-13-acetate exposure enhances Adriamycin's cytotoxicity as well as its ability to provoke DNA damage (measured by alkaline elution). Likewise, down-regulation of PKC by extended 12-O-tetradecanoylphorbol-13-acetate exposure partially protects the cells from Adriamycin-induced cytotoxicity as well as from DNA damage. Thus, the ability of cells to be injured by Adriamycin appears to be correlated with the activity of PKC. Multidrug-resistant subline Sarcoma 180A10 cells have the same total quantity of membrane-recruitable PKC as the sensitive parent Sarcoma 180 cells, as determined by [3H]phorbol-12,13-dibutyrate binding. However, the resistant cells have a significantly higher intrinsic PKC activity and an altered ability to translocate the enzyme to the cell surface. Taken together, the results raise the possibility that cell signaling mechanisms, particularly those involving protein kinase C, may play an important role in mediating the biological action of the anticancer drug Adriamycin.
Insights
Adriamycin rapidly increases diacylglycerol and protein kinase C (PKC) activity in cancer cells, influencing cytotoxicity. This suggests PKC signaling is crucial for Adriamycin
Area of Science:
- Cell Biology
- Pharmacology
- Biochemistry
Background:
- Adriamycin is an anticancer drug with diverse cellular effects.
- Cytotoxicity is a key mechanism of Adriamycin action.
- Early cellular alterations induced by Adriamycin are not fully understood.
Purpose of the Study:
- To investigate the early cellular events following Adriamycin exposure.
- To explore the role of diacylglycerol and protein kinase C (PKC) in Adriamycin's effects.
- To determine if PKC activity correlates with Adriamycin-induced cytotoxicity and DNA damage.
Main Methods:
- Exposure of Sarcoma 180 cells to Adriamycin.
- Measurement of diacylglycerol production.
- Assay of cytosolic and membrane-bound protein kinase C (PKC) activity.
- Experiments using phorbol ester (12-O-tetradecanoylphorbol-13-acetate) to modulate PKC.
- Assessment of DNA damage using alkaline elution.
- Analysis of PKC in multidrug-resistant cell lines.
Main Results:
- Adriamycin rapidly elevates diacylglycerol production within 10 minutes.
- Cytosolic PKC activity significantly increases (56%) post-Adriamycin exposure.
- PKC activation by phorbol ester enhances Adriamycin cytotoxicity and DNA damage.
- PKC down-regulation partially protects cells from Adriamycin's effects.
- Multidrug-resistant cells show altered PKC activity and translocation.
Conclusions:
- Increased diacylglycerol and PKC activity are early events in Adriamycin-treated cells.
- PKC signaling appears to play a significant role in mediating Adriamycin's cytotoxic effects.
- Altered PKC activity may contribute to multidrug resistance to Adriamycin.