The Effects of Sars-Cov-2 Infection on Cancer-Like Phenotypes and Cytokine Production in Cancer Cell Line

Abstract

The occurrences of cancer and cancer-related mortality are a growing burden worldwide and co infections with other pathogens such as viruses might contribute to disease pathogenesis. Cancer is one of the leading comorbidities for the recent global pandemic ‘Coronavirus Disease 2019’ (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The infection causes severe respiratory injury, organ failure, and hyper-production of inflammatory cytokines. The causative virus initiates infection by attaching to the human Angiotensin Converting Enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) within the viral spike protein. Also, successful infection is dependent on the activation of the spike by host proteases like the Transmembrane serine protease 2 (TMPRSS2). Studies have shown that ACE2 and TMPRSS2 are present in healthy cells and may play important roles in regulating cellular function. Other studies have also shown aberrant ACE2 expression in cancer cells. This research sought to study how ACE2 expressed on cancer cells interacts with SARS-CoV-2 spike protein and pseudoviruses (PV’s), and how this interaction affects cancer phenotypic properties and cytokine expression. Gene expression analysis for ACE2 and TMPRSS2 in breast, colorectal, and prostate tumors tissues and normal tissues was carried out using Gene Expression Profiling Interactive Analysis (GEPIA) software. The protein expression levels of ACE2 on seven cancer cell lines (MDA-MB-231, MDA-MB-468, DLD-1, COLO205, HCT-15, 22RV1, and BPH1) were screened using dot blot assay. In vitro, analysis of the interaction of Spike and ACE2 was examined by producing SARS-CoV-2 pseudovirus (PV) to infect the cell lines. The transduction efficiency of the PVs was measured by the quantification of luciferase activity. Cancer cell proliferation, viability, migration, and angiogenic markers were analyzed post-infection using3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, cell titer Glo viability assay, wound healing assay, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cytokines expression post-infection was investigated using qRT-PCR. The effects were further confirmed with live virus (LV) infections using MTT and qRT-PCR. Using in silico analysis, ACE2 was shown to be expressed highly in colorectal tissues and low in breast and prostate tissues. In vitro, ACE2 expression data was consistent with the in-silico analysis. However, out of 7 cell lines, a significant PV infection was observed only in 22RV1 (prostate) and DLD-1 (colorectal). PV decreased 22RV1 proliferation but have an inconclusive effect on DLD 1. Proliferation and migration were increased in LV- infected 22RV1 but were decreased in LV infected DLD-1. Infections increased cytokine levels in 22RV1. Similarly, PV increased cytokine expression for DLD-1 cells. However, LV downregulated IL-1β and IL-8 in DLD-1. LV infection affected the expression of genes involved in proliferation, migration, angiogenesis, and the expression of cytokines. Although ACE2 expression might not ensure possible infection of SARS CoV-2. The current findings suggest that infection of 22RV1 and DLD-1 with the virus may affect their cellular properties and gene expression. The infection can upregulate and downregulate the expression of cytokines in cancer contributing to the progression or regression of these cancers. Additionally, PV might not be an appropriate model for the study on the viral-host response partly because LV infections and PV infections have contrasting effects on cancer properties in different cell lines.