Upregulation of gap junctional intercellular communication and connexin 43expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells

Purpose: Downregulation of gap junctional intercellular communication (GJIC ) has been implicated in carcinogenesis. This is a result of altered expres sion of connexins, the proteins that mediate GJIC, including connexin 43 (C x43). Our aim was to evaluate the effect of known inducers of Cx43 on the c hemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherape utic agents. Methods: We examined the effect of dibutyryl-cyclic AMP (db-cA MP) and all-tr ans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma -glutamyl-cysteine-synthetase (gamma -GCS) levels, and glutathion e S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxor ubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db -cAMP and/or tRA. Results: Exposure to db-cAMP led to the upregulation of G JIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylatio n were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Tre atment with db-cAMP, but not with tRA, was associated with a significant in crease in the cytotoxic effects of the anticancer drugs doxorubicin, melpha lan and BCNU as shown by a decrease in their IC50 values. Concomitant expos ure to db-cAMP and tRA, however, had a more pronounced effect on cell sensi tization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma -GCS activities remained unchang ed. Conclusions: This study suggests that GJIC plays a role in cellular dru g resistance, and highlights the potential use of GJIC modulators in combin ation with chemotherapy. Also, this is the first study exploring the abilit y of both db-cAMP and tRA to enhance cell chemosensitivity.