Silver nanoparticles trigger proteotoxicity in multiple myeloma cells via a mechanism distinct from bortezomib and remain efective in BTZ-resistant myeloma Free

Background: Multiple myeloma (MM), characterized by abnormal clonal plasma cell expansion, is the second most common hematologic malignancy accounting for 13% of all cases. Proteosome inhibitors (PIs), such as bortezomib (BTZ), are commonly included as part of first line of treatment of MM. Due to dose-limiting toxicity and other factors, acquired resistance to PIs frequently occurs in MM patients, and those who become refractory to PIs have significantly worse outcomes. Silver nanoparticles (AgNPs) induce cancer cell death through increased protein aggregation leading to proteotoxic stress and cell death. MM cells are exquisitely sensitive to proteotoxicity due to extensive synthesis and secretion of abnormal immunoglobins, and we hypothesized that AgNPs might provide a new therapeutic option for this disease. The current study compares the efficacy and mechanisms of action of BTZ and AgNPs as single agents and combined in BTZ-sensitive and BTZ-resistant MM cells. The goal of this research project is to develop more effective, less toxic treatments that could exploit known vulnerabilities in MM and overcome resistance to current treatments.

Methods: The IC₅₀ values for treatment of JJN3, U266B1, MM1R, MM1S, KMS-12-BM parent, and KMS-12-BM BTZ-resistant MM cells with AgNP or BTZ were determined. Proteostat staining was used to confirm protein aggregation following AgNP and BTZ treatments. Time and dose-dependent effects of AgNPs and BTZ on protein ubiquitination and autophagy/autophagic flux were quantified by western blot using antibodies for ubiquitin, LC3I, LC3II, and p62. AgNP and BTZ synergy studies were conducted through fixed-ratio combination dosing (Chou-Talay method). KMS-12-BM parent and KMS-12-BM-BTZ-resistant MM cell lines were used to evaluate AgNP efficacy and mechanism of action in the context of BTZ resistance.

Results: Both AgNPs and BTZ were effective against MM cells at low doses [AgNPs: 6-18 µg/mL AgNPs; BTZ: 2-8 nM]. The IC₅₀ for BTZ was 4-fold higher in the KMS-12-BM BTZ-resistant cells than KMS-12-BM parent cells [25 nM vs. 6nM], but both cell lines had a similar IC₅₀s to AgNP [6 µg/mL]. When combined, AgNPs and BTZ showed synergy (combination index of 0.13; dose enhancement ratio of 15). Both AgNPs and BTZ, given at doses near their IC₅₀ concentrations, increased protein aggregation in all MM cells but had distinct effects on the ubiquitin-proteasome system and autophagy. Increased ubiquitination was observed following AgNP and BTZ treatment in all cell lines. However, accumulation initially was greater in AgNP treated cells after 3 h, but after 18 h was greater in BTZ treated cells. BTZ did not induce autophagy in BTZ-sensitive MM cell lines at any time point at doses near the IC₅₀. In contrast, AgNPs induced autophagy as indicated by increased LC3II:LC3I and decreased p62 in 5 of the 6 MM cell lines tested. KMS-12-BM BTZ resistant cells had higher baseline levels of LC3I and LC3II than parental cells. When treated with BTZ, increased ubiquitination was observed in both the KMS-12-BM BTZ-resistant and parental cells after 3 h, but the increase was transient in the BTZ-resistant cells. BTZ induced autophagy in KMS-12-BM BTZ-resistant cells at doses near its IC₅₀, but autophagy was only observed in parental cells when they were treated with BTZ at doses 4-8 times the IC₅₀. AgNPs increased autophagy in KMS-12-BM BTZ-resistant cells, but not parental cells when treated with identical doses of AgNPs (IC₅₀s were similar for both cell lines).

Conclusion: Both AgNPs and BTZ induce proteotoxicity in MM. AgNPs and BTZ induced synthetic lethality when used in combination, and BTZ resistance does not alter sensitivity to AgNPs. Mechanistically, AgNPs cause more rapid accumulation of ubiquitinated proteins compared to BTZ, but sustained accumulation of ubiquitinated proteins is only observed in BTZ treated cells. The baseline levels of autophagy are higher in BTZ-resistant cells compared to parental cells. Most MM cells (BTZ-sensitive and resistant) activate autophagy in response to AgNPs. In contrast, only BTZ-resistant cells activate autophagy in response to therapeutic doses of BTZ.Taken together, these data indicate AgNPs work through a distinct mechanism from BTZ. Upregulation of autophagy protects against BTZ, but not AgNP-induced proteotoxicity. As such, AgNPs are a promising therapeutic option for overcoming BTZ resistance and show potential as a dose-sparing adjunct in combination with BTZ.