Combined Cytotoxic Effect of Inhibitors of Proteostasis on Human Colon Cancer Cells.

Despite significant progress in the diagnosis and treatment of colorectal cancer, drug resistance continues to be a major limitation of therapy. In this regard, studies aimed at creating combination therapy are gaining popularity. One of the most promising adjuvants are inhibitors of the proteostasis system, chaperone machinery, and autophagy. The main HSP regulator, HSF1, is overactivated in cancer cells and autophagy sustains the survival of malignant cells. In this work, we focused on the selection of combination therapy for the treatment of rectal cancer cells obtained from patients after tumor biopsy without prior treatment. We characterized the migration, proliferation, and chaperone status in the resulting lines and also found them to be resistant to a number of drugs widely used in the clinic. However, these cells were sensitive to the autophagy inhibitor, chloroquine. For combination therapy, we used an HSF1 activity inhibitor discovered earlier in our laboratory, the cardenolide CL-43, which has already been proven as an auxiliary component of combined therapy in established cell lines. CL-43 effectively suppressed HSF1 activity and Hsp70 expression in all investigated cells. We tested the autophagy inhibitor, chloroquine, in combination with CL-43. Our results indicate that the use of an inhibitor of HSF1 activity in combination with an autophagy inhibitor results in effective cancer cell death, therefore, this therapeutic approach may be a promising treatment regimen for certain patients.

Prevention of High Glucose-Mediated EMT by Inhibition of Hsp70 Chaperone.

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.