Rintatolimod Induces Antiviral Activities in Human Pancreatic Cancer Cells: Opening for an Anti-COVID-19 Opportunity in Cancer Patients?

Abstract

Severe acute respiratory virus-2 (SARS-CoV-2) has spread globally leading to a devastating loss of life. Large registry studies have begun to shed light on the epidemiological and clinical vulnerabilities of cancer patients who succumb to or endure poor outcomes of SARS-CoV-2. Specific treatment for COVID-19 infections in cancer patients is lacking while the demand for treatment is increasing. Therefore, we explored the effect of Rintatolimod (Ampligen^®) (AIM ImmunoTech, Ocala, FL, USA), a Toll-like receptor 3 (TLR3) agonist, to treat uninfected human pancreatic cancer cells (HPACs). The direct effect of Rintatolimod was measured by targeted gene expression profiling and by proteomics measurements. Our results show that Rintatolimod induces an antiviral effect in HPACs by inducing RNase-L-dependent and independent pathways of the innate immune system. Treatment with Rintatolimod activated the interferon signaling pathway, leading to the overexpression of several cytokines and chemokines in epithelial cells. Furthermore, Rintatolimod treatment increased the expression of angiogenesis-related genes without promoting fibrosis, which is the main cause of death in patients with COVID-19. We conclude that Rintatolimod could be considered an early additional treatment option for cancer patients who are infected with SARS-CoV-2 to prevent the complicated severity of the disease.

Keywords: RNase L; Rintatolimod; SARS-CoV-2; Toll-like receptor 3; cytokines; epithelial cancerous cells; interferon signaling.

Figure 1

Rintatolimod stimulated interferon production and activated the interferon signaling pathway in HDAC cells. Heat maps of genes involved in the interferon signaling pathway. The linear values of the gene expression were used to generate the heat maps. The expressions were gene-wise scaled and are displayed as colors ranging from blue (high expression) to yellow (low expression) as shown in the key. Cells were treated with various concentrations of Rintatolimod displayed as the intensity of green color from dark green (high dose) to white (PBS control).

Figure 2

Rintatolimod produced immunomodulatory activity in HPAC cells. Heat maps of cytokines and interleukins that were differentially regulated as an effect of Rintatolimod. The expressions were gene-wise scaled and are displayed as colors ranging from blue (high expression) to yellow (low expression) as shown in the key. Cells were treated with various concentrations of Rintatolimod displayed as the intensity of green color from dark green (high dose) to white (PBS control).

Figure 3

Rintatolimod induced the expression of MHC class I and II histocompatibility in HPDA cells. Heat maps of HLA-genes that were differentially regulated as an effect of Rintatolimod. The expressions were gene-wise scaled and are displayed as colors ranging from blue (high expression) to yellow (low expression) as shown in the key. Cells were treated with various concentrations of Rintatolimod displayed as the intensity of green color from dark green (high dose) to white (PBS control).

Figure 4

Schematic presentation of the overexpressed genes and the antiviral pathways activated by Rintatolimod. Rintatolimod (Ampligen; dsPoly (I: C12U)) enters the cells and targets the TLR3 in the endosome to start a cascade of actions. A—Stimulation of TLR3, which activates TICAM1 and TRIF3 that are involved in innate immunity against pathogens. Activation of TICAM1 and MyD88 mediate NF-kB expression that subsequently activates transcription factors such as IRF1 and IRF7. Interferon Regulatory Factors activate STAT1, 2, and 3 that stimulate the innate and acquired immune responses, mediate cellular responses to interleukin, and regulate inflammatory responses to infection. B—Activation of STAT1 and 2 upregulate the expression of OAS3, which induces the synthesis of 2′,5′-oligoadenylate (2–5 A) from ATP upon binding of dsRNA. 2–5 A activates RNase L, causing endonucleolytic cleavage of viral ssRNAs and blocking virus replication. C—TLR3 activation induces the expression of members of the IFIT family. The upregulation of IFIT1 and IFIT2 result in restriction of ssRNA virus replication in the cytosol. In addition, the IFIT family restricts virus replication through an alternation of protein synthesis and the ability of an antiviral protein to bind viral RNA directly. D—Activation of STAT 1, 2, and 3 activate the expression of interferon stimulatory genes (ISGs) like ISG15, which induces the antiviral state in the cytosol. ISG15 acts on ssRNA and directly degrades viral RNA. Treatment with Rintatolimod increased the expression of IFNAR1 and IFNAR2, which recognize interferon and activate JAK/STAT signaling pathways. E—Activation of STAT signaling pathways leads to the production of various cytokines, which results in the activation of the adaptive immune system. The pathway image was created with biorender.com.

Figure 5

Treating cancer cells with Rintatolimod did not upregulate genes involved with fibrosis. The expression of TLR3 (A) was upregulated in CFPAC1 cells after treating with Rintatolimod. The expression of VCAM1 (B) and TGFβ2 (D) was upregulated by Rintatolimod. However, no upregulation of TGFβ1 (C) was observed after treating with Rintatolimod in both cell lines.