pd-150606 and Melanoma

Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ΔPK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ΔPK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ΔPK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance.

Topics: Acrylates; Adaptor Proteins, Signal Transducing; Apoptosis; Autophagy; Calpain; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Neoplastic Stem Cells; Oncolytic Virotherapy; Oncolytic Viruses; Sequestosome-1 Protein; Spheroids, Cellular; Virus Replication

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Topics: Acrylates; Butanes; Calpain; Cell Cycle; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Humans; I-kappa B Proteins; Melanoma; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Protease Inhibitors; Proteasome Inhibitors