Examination of the effects of HIV protease inhibitors (HIV-PIs) on the function of the novel s-DAPK-1 in Human Papilloma Virus (HPV)-related cervical cancers

Abstract

Cervical cancer treatment continues to be every country’s nightmare due to ineffectiveness and non-specificity of the current therapeutic options, late diagnosis and chemo drug resistance. An escalating resistance of cervical cancer cells to chemotherapy coupled with severe side effects of commonly used cytotoxic drugs has intensified the need to search for new anti-cancer agents. Several drugs initially approved for non-cancerous conditions have recently been found to possess cytostatic effects on cancer cells. Thus, these drugs could be expediently repurposed for use as anti-cancer agents because they have already been tested for safety in animals and humans. In light of this, this study sought to investigate the possibility of adapting pure HIV protease inhibitors (HIV-PIs) and their over-the counter tablets for anti-cervical cancer therapeutic purposes. Additionally, since cervical cancer is viewed as a pathology that is partly driven by genes, it was of interest to understand the expression of short-DAPK-1 known as s-DAPK-1, which remains unexplored in cervical cancer, and the HIV-PI s' mechanisms of action. Therefore, this study was aimed at investigating the effect of HIV-PIs on s-DAPK-1 and other cancer-related genes in HPV-induced cervical cancer cells. To address the aim of this study, the MTT viability and Muse™ Count & Viability assays were used to evaluate the effect of the pure HIV protease inhibitors and their tablet forms on the viability of CaSki and HeLa cervical cancer cell lines, as well as on the non-cancerous cells (HEK-293). To detect the mode of death induced by pure HIV-PIs (lopinavir and atazanavir) and their tablet forms (Aluvia and Ritoataz) in HPV-associated cervical cancer cells, apoptosis was assessed using the Annexin V Assay. Apoptosis-related proteins regulated by HIV-PIs and their tablet forms were detected using the Human Apoptosis Array profiler. In addition, the Muse™ Cell Cycle assay was used to assess the effect of HIV-PIs and their tablet forms on cell cycle progression of the cervical cancer cells. The Polymerase Chain Reaction (PCR) was used to determine the expression of s-DAPK-1 in HIV-PIs-treated and untreated cervical cancer cells, and to elucidate the effect of pure HIV protease inhibitors and HIV protease inhibitor tablets on its expression. In addition to the use of PCR, the proteome profiler human apoptosis array kit was used to detect other cellular targets of pure HIV-PIs and their over-the counter tablets in HPV-associated cervical cancer cells. Furthermore, various bioinformatics tools such as ProtScale, ProteinPrompt, I-TASSER, PSIPRED, ProtParam, ScooP, Hawkdock, Phyre2, SAVES and PROCHECK along with user-friendly databases such as NCBI, TarBase and Protein Data Bank (PDB) were used to understand s-DAPK-1 regulation, 3D structure, physicochemical and thermodynamic properties. This study demonstrated that lopinavir and atazanavir pure HIV-PIs, as well as Aluvia and Ritoataz tablets did not affect the viability of non-cancerous cells (HEK-293), confirming the safety of the HIV-PIs. However, they have significantly decreased the viability of the CaSki and HeLa cervical cancer cells in a dose- and time-dependent manner. It is important to note that, relative to HeLa cells, a higher concentration of lopinavir (IC50=150 μM) and Ritoataz (IC50=180 μM) was required to reduce the viability of CaSki cells by 50%. All the HIV-PIs triggered apoptosis in CaSki and HeLa cervical cancer cells. However, lopinavir (39.925±1.483 in CaSki and 41.583±1.001 in HeLa) and atazanavir (49.092±0.9376 in CaSki and 36.717±1.729 in HeLa) significantly (p ≤ 0.001) exhibited the highest induction of apoptosis. In contrast, the corresponding tablets of Aluvia (24.418± 2.346 in CaSki and 26.795±0.6805 in HeLa) and Ritoataz (25.310±1.323 in CaSki and 28.432±2.374 in HeLa) induced a significantly (p ≤ 0.001) lower levels of apoptosis. In addition, pure HIV protease inhibitors along with their tablet forms significantly (p ≤ 0.05) regulated the activity of various apoptosis-related proteins, including phosphorylation p53 (S392) and Rad17 (S635). The HIV-PIs upregulated SMAC/Diablo, and Bcl-2, suggesting induction of an intrinsic apoptosis pathway, with cervical cells resisting cell death by upregulating Bcl-2. The s-DAPK-1 variant was significantly downregulated in HeLa cells relative to non-cancerous HEK-293 cells, suggesting that it may be a tumour suppressor. In addition, pure HIV protease inhibitors and the HIV protease inhibitor tablets did not influence the expression of s-DAPK-1 in cervical cancer cells. Furthermore, the in-silico approach, to determine s-DAPK-1 regulation, successfully identified several s-DAPK-1-specific microRNAs. In addition, phyre2 database demonstrated that the s-DAPK-1 isoform possesses 40% alpha helices and 4% beta strands, forming a stable 3D structure. Moreover, s-DAPK-1 was discovered to withstand high temperatures and to interact with a variety of proteins involved in tumour progression and gene regulation, including Prion protein and Histone H2B type 2-E (H2B2E). The findings of this study highlight the HIV protease inhibitors as promising anticancer agents, demonstrating significant effects on inducing cell death and suppressing proliferation. Furthermore, this work has discovered more anticancer drug targets that should be exploited for drug development. Moreover, since this is the first study to explore the expression and regulation of s-DAPK-1 by therapeutic agents, there is a pressing need to identify novel compounds that can modulates s-DAPK-1 and to explore its potential tumour suppressor function in cancer cells.