Physical plasma-triggered ROS induces tumor cell death upon cleavage of HSP90 chaperone

Abstract

HSP90 is a ubiquitously expressed molecular chaperone implicated in the correct folding and maturation of a plethora of proteins including protein kinases and transcription factors. While disruption of chaperone activity was associated with augmented cancer cell death and decreased tumor growth both in vitro and in vivo, the regulation of HSP90 is not clearly understood. Here we report that treatment of cancer cells with cold physical plasma, an emerging and less aggressive tumor therapy, resulted in ROS generation which subsequently triggered the cleavage of HSP90. Notably, cleavage of HSP90 was followed by the degradation of PKD2, a crucial regulator of tumor growth and angiogenesis. Pre-sensitization of cancer cells with subliminal doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, followed by treatment with cold-plasma, synergistically and negatively impacted on the viability of cancer cells. Taken together, cold-plasma can be used in conjunction with pharmacologic treatment in order to target the expression and activity of HSP90 and the downstream client proteins implicated in various cancer cell capabilities.

Figure 1

(A,B) The atmospheric pressure argon plasma jet kINPen and principle of reactive species generation. (C) Representative images of redox-sensitive dye CM-H₂DCF-DA-loaded MDA-MB-231 cells in control and plasma-treated samples. (D) Quantitative image analysis of mean fluorescence intensity of the CM-H₂DCF-DA redox-sensitive dye. Plasma treatment time was 60 seconds, image acquisition was performed 1 hour after plasma treatment.

Figure 2

Cold plasma treatment results in impaired cancer cell viability and augmented apoptosis. (A) Representative image of resazurin assay, plasma treatment reduced transformation to pink resofurin indicated decrease in viability 24 hours after plasma treatment. (B) Viability of SW480, MDA-MB-231, and Capan1 in response to plasma treatment. MDA-MB-231 cells were most sensitive, followed by SW480, and Capan1, respectively. For each cell line, statistical coimparison (one.way analysis of variance with Dunnett post-test) of each plasma treatment time (60 s, 120 s, 180 s) compared to argon gas control (180 s) revealed significant (p < 0.01 or p < 0.001) differences. (C) Twenty-four hours following argon gas or plasma treatment (both 60 s), lysates of MDA-MB-231 and SW480 were subjected to SDS-PAGE followed by incubation with PARP antibodies. β-actin was used as loading control.

Figure 3

Cold plasma treatment is associated with cleavage of HSP90 chaperone and PKD2 degradation. (A–D) Various cancer cell lines were subjected to treatment with plasma jet (60 s). Twenty-four hours later, cleared lysates were used for western blot analysis in order to determine the HSP90 and PKD2 abundance.

Figure 4

Cold plasma treatment boosts cell death in drug pre-sensitized cancer cells. (A) MDA-MB-231 breast and (B) SW480 colon cancer were treated for 24 hours with HSP90 inhibitor as indicated. Apoptosis was determined by examining the levels of cleaved PARP in western blot analysis. (C,E) MDA-MB-231 and (D,F) SW480 cancer cells were either incubated with 50 nM PU-H71 or subjected to plasma treatment (60 s) or both. A synergistic toxicity between PU-H71 and plasma treatment was observed at 24 h. RFU = relative fluorescence units.

Figure 5

Ectopic PKD2 expression is not sufficient to restore cancer cell viability after treatment with cold plasma. Cancer cells were transduced with a PKD2 expression (PKD2 o.e.) or empty vector (e.v.). After selection with antibiotic, lysates of transduced cancer cells were prepared and SDS-PAGE was conducted with PKD2 antibody. β-actin was used as loading control. (B,C) MDA-MB-231 breast and SW480 colon cancer cells (e.v. as well as PKD2 o.e.) were treated with plasma (60 s) and/or 50 nM PU-H71, and cell viability was determined at 24 h.

Figure 6

Cleavage of HSP90 and degradation of PKD2 following cold plasma treatment is associated with cancer cell death. Physical plasma treatment- generated ROS is followed by HSP90 cleavage and subsequent destabilization and degradation of PKD2. While PKD2 degradation plays an important role in cancer cell death, additional essential molecules such as STK33, also contribute to the apoptotic event. Furthermore, pre-treatment of cancer cells with subliminal doses of HSP90 inhibitor followed by cold plasma treatment boosts cell death in human cancer.