Abstract

   Until today, the coronavirus disease 2019 (COVID-19) pandemic has
   caused 6,043,094 deaths worldwide, and most of the mortality cases have
   been related to patients with long-term diseases, especially cancer.
   Autophagy is a cellular process for material degradation. Recently,
   studies demonstrated the association of autophagy with cancer
   development and immune disorder, suggesting autophagy as a possible
   target for cancer and immune therapy. Laminarin is a polysaccharide
   commonly found in brown algae and has been reported to have
   pharmaceutic roles in treating human diseases, including cancers. In
   the present report, we applied network pharmacology with systematic
   bioinformatic analysis, including gene ontology (GO) enrichment, Kyoto
   Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, reactome
   pathway analysis, and molecular docking to determine the pharmaceutic
   targets of laminarin against COVID-19 and cervical cancer via the
   autophagic process. Our results showed that the laminarin would target
   ten genes: CASP8, CFTR, DNMT1, HPSE, KCNH2, PIK3CA, PIK3R1, SERPINE1,
   TLR4, and VEGFA. The enrichment analysis suggested their involvement in
   cell death, immune responses, apoptosis, and viral infection. In
   addition, molecular docking further demonstrated the direct binding of
   laminarin to its target proteins, VEGFA, TLR4, CASP8, and PIK3R1. The
   present findings provide evidence that laminarin could be used as a
   combined therapy for treating patients with COVID-19 and cervical
   cancer.

   Keywords: functional foods, laminarin, cervical carcinoma, COVID-19,
   biotargets

Introduction

   Since the outbreak of COVID-19 in 2019, there have been over
   456,797,217 confirmed cases of COVID-19 worldwide, leading to 6,043,094
   deaths, as reported to the World Health Organization (WHO) (14 March
   2022). The mortality rate of the latest variant, omicron, is relatively
   low, but its high infection rate continues to be a pressing problem in
   our society ([37]Bouzid et al., 2022). Moreover, the mortality rate is
   very high in patients with long-term diseases, especially cancer
   patients ([38]Aghamirza Moghim Aliabadi et al., 2022). Therefore, there
   is an urgent need to identify alternative drug or compound to help
   patients with COVID-19 and cancer. Cervical carcinoma is a common
   malignant tumor that occurs in the female reproductive system
   ([39]Cohen et al., 2019). According to the data from the WHO, cervical
   carcinoma is the fourth most common cancer among women, leading to
   342,000 deaths in 2020 globally
   ([40]https://www.who.int/news-room/fact-sheets/detail/cervical-cancer).

   Autophagy is a mechanism for cellular material degradation that occurs
   under stressful conditions ([41]Yun and Lee, 2018). Cumulating studies
   showed that autophagy disorder is closely associated with the
   development of cancer ([42]Levy et al., 2017). In cervical cancer,
   autophagy-related genes were reported to predict the prognosis of
   cervical cancer ([43]Jiang et al., 2021). In vitro and in vivo
   protective autophagic mechanisms resulted in antitumor activity in
   cervical cancer ([44]Zhang F. et al., 2022). On the contrary, autophagy
   was found to interplay with the antiviral IFN-I response, suggesting
   the targeting of autophagy as a potential target and strategy for
   treating COVID-19 ([45]Bonam et al., 2020; [46]García-Pérez et al.,
   2020). Therefore, targeting autophagy could be a potential option for
   treating patients with COVID-19 and cervical cancer.

   Laminarin is a polysaccharide commonly found in brown algae ([47]Chen
   et al., 2021). Recent studies suggested the pharmaceutic roles of
   laminarin in human diseases, including cancers. A study of colorectal
   carcinoma cells demonstrated the combined anticancer effect of sulfated
   laminarin ([48]Malyarenko et al., 2021). Zhang’s group also suggested
   that laminarin sulfate can be used as a carrier to inhibit heparinase
   and treat melanoma lung metastasis ([49]Zhang et al., 2021). Besides
   treating cancer, laminarin was reported to regulate IL-10 production
   and host immunogenicity ([50]Ferreira et al., 2007; [51]Korotchenko et
   al., 2021). In this report, we applied network pharmacology with
   bioinformatics analysis, including gene ontology (GO) enrichment, Kyoto
   Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and
   molecular docking, to investigate the pharmaceutic targets of laminarin
   against COVID-19 and cervical cancer through autophagy. The findings of
   the present study provide evidence that laminarin could be used as a
   combined therapy for treating patients with COVID-19 and cervical
   cancer.

Materials and methods

Identification of laminarin-, cervical carcinoma-, COVID-19-, and
autophagy-associated genes

   The mRNA expression profile of cervical carcinoma (CC) was obtained
   upon searching the dataset of [52]GSE63514 from the Gene Expression
   Omnibus (GEO) database, which included 24 normal and 28 cervical
   cancers ([53]Barrett et al., 2013). The differentially expressed genes
   (DEGs) of cervical carcinoma were analyzed by the GEO2R tool ([54]den
   Boon et al., 2015). The genes with |log[2] fold change| > 1 and
   −log[10]p-value > 1.3 were considered DEGs. For the identification of
   COVID-19- and autophagy-associated genes, the online databases
   including the GeneCards database ([55]Stelzer et al., 2016), Online
   Mendelian Inheritance in Man (OMIM) database ([56]Hamosh et al., 2002),
   and National Center for Biotechnology Information (NCBI) were accessed
   ([57]National Library of Medicine National Center for Biotechnology
   Information (NCBI), 2022). The pharmaceutical targets of laminarin were
   harvested using online databases, including Comparative Toxicogenomics
   Database (CTD) ([58]Davis et al., 2020), Swiss Target Prediction
   database ([59]Daina et al., 2019), SuperPred database ([60]Nickel et
   al., 2014), and PharmMapper ([61]Wang X. et al., 2017). The target
   genes were subjected to UniProt Knowledgebase (UniProtKB) for human
   database correction ([62]The UniProt Consortium, 2021). To further
   determine the targets of laminarin against cervical carcinoma and
   COVID-19 through autophagy, the laminarin-, CC-, COVID-19- and
   autophagy-associated genes and targets were overlapped. The common
   genes were considered the possible targets.

Core target screening and network analysis

   The common genes were subjected to Bioconductor packages, including
   ClusterProfiler and ggplot2 tools, to analyze and visualize the
   biological processes and pathways controlled by the targets of
   laminarin against cervical carcinoma and COVID-19 through autophagy.
   ClusterProfiler provides GO enrichment, KEGG enrichment analysis, and
   Reactome pathway analysis ([63]Jassal et al., 2020; [64]Wu T. et al.,
   2021). Ggplot2 was used to visualize the enrichment results. The terms
   with adjusted p-value < 0.05 was considered statistically significant.
   The targets were used for protein–protein interaction (PPI) analysis
   using the STRING platform, and the confidence score was 0.4
   ([65]Szklarczyk et al., 2021). The NetworkAnalyzer of Cytoscape
   software was also used to integrate the result of targets
   identification, GO, and KEGG ([66]Shannon et al., 2003).

Molecular docking

   The binding affinity of laminarin with its predicted targets was
   assessed using molecular docking. The chemical structure of laminarin
   was obtained from the PubChem database ([67]Wang Y. et al., 2017). The
   protein structure of VEGFA, TLR4, and CASP8 was obtained from the
   Protein Data Bank (PDB) ([68]Berman et al., 2000). The ChemBio3D Draw
   tool of ChemBioOffice 2010 software was used to download
   three-dimensional structures of these proteins, followed by the MM2
   energy optimization. The PDB file was converted to the pdbqt file
   format using AutoDock Tools 1.5.6 of the AutoDock Vina software (that
   can be recognized by the AutoDock Vina program for ligand docking
   program) ([69]Trott and Olson, 2010). The docking parameter was
   determined according to the size of the root-mean-square deviation
   (RMSD) between the docked ligand molecule and the original ligand
   molecule. An RMSD of less than 4 Å was the threshold for the
   conformation of the ligand for matching the conformation of the
   original ligand in the molecular docking analysis.

Results

Identification of laminarin’s targets against cervical carcinoma and COVID-19
through autophagy

   Upon analysis of the [70]GSE63514 dataset of GEO, we identified 3,169
   DEGs, including 1,093 upregulated genes and 2,076 downregulated genes
   in cervical carcinoma ([71]Figure 1A). By searching the databases, we
   obtained 1,390 COVID-19-associated genes ([72]Figure 1B). In addition,
   we found 284 pharmaceutical targets of laminarin and 1,744
   autophagy-associated genes ([73]Figure 1B). The identified gene and
   targets were overlapped to determine the possible targets of laminarin
   against cervical carcinoma and COVID-19 through autophagy. Ten common
   targets (CASP8, CFTR, DNMT1, HPSE, KCNH2, PIK3CA, PIK3R1, SERPINE1,
   TLR4, and VEGFA) were observed, and the PPI analysis showed their
   interaction ([74]Figure 1B).

FIGURE 1.

   [75]FIGURE 1
   [76]Open in a new tab

   Identification of laminarin’s targets against cervical carcinoma and
   COVID-19 through autophagy. (A) Volcano plot showed the differential
   gene expression in cervical carcinoma. Genes with |log[2] fold change|
   > 1 and −log[10] p-value > 1.3 were considered differentially expressed
   genes. Green dots represented downregulated genes, and red dots
   represented upregulated genes. (B) Left panel: Venn diagram showed the
   number of overlapped laminarin-, COVID-19-, cervical carcinoma-, and
   autophagy-associated genes. Right panel: protein–protein interaction of
   the laminarin-, COVID-19-, cervical carcinoma-, and
   autophagy-associated genes.

Laminarin’s targets were involved in the cell death and immune responses

   To understand the biological roles controlled by laminarin targets, GO
   enrichment analysis was applied. The result of GO analysis highlighted
   the biological processes related to cell proliferation and
   differentiation ([77]Figure 2A), cell death such as extrinsic apoptotic
   signaling pathway, TRAIL-activated apoptotic signaling pathway, and
   regulation of necroptotic process ([78]Figure 2B). In addition,
   laminarin’s targets played roles in immune responses such as regulation
   of innate immune response, regulation of leukocyte differentiation,
   macrophage differentiation, B-cell activation, and production of
   interleukin ([79]Figure 2C). Furthermore, many kinase activities such
   as regulation of MAP kinase activity, regulation of I-kappaB
   kinase/NF-kappaB signaling, regulation of p38MAPK cascade, and protein
   kinase C signaling were highlighted in our result ([80]Figure 2D). In
   terms of molecular function, laminarin’s targets contributed to
   different binding and ion channel activities, especially the functions
   related to immune functions such as cytokine receptor binding, tumor
   necrosis factor receptor binding, and chemoattractant activity
   ([81]Figure 2E). These biological processes and molecular functions
   were suggested to occur in different cellular components, including
   platelet alpha granule lumen, ion channel complex, cytoplasmic vesicle
   lumen, perinuclear endoplasmic reticulum, pericentric heterochromatin,
   transmembrane transporter complex, transporter complex, early endosome,
   phagocytic cup, and phosphatidylinositol 3-kinase complex ([82]Figure
   2F).

FIGURE 2.

   FIGURE 2
   [83]Open in a new tab

   Involvement of laminarin’s targets in cell death and immune responses.
   Gene ontology enrichment analysis of laminarin’s targets highlighted
   the biological processes related to (A) cell proliferation and
   differentiation, (B) cell death, (C) immune responses, and (D) kinase
   activities. (E) Laminarin’s targets contributed to molecular functions,
   including binding and ion channel activities. (F) Laminarin’s targets
   played roles in different cellular components. The size of a bubble
   represents the number of genes, and the color of the bubble represents
   the significance of the terms.

Laminarin’s targets contributed to the pathways related to cancer and viral
infection

   To further investigate the signaling pathways controlled by laminarin’s
   targets, KEGG enrichment analysis, and Reactome pathway analysis were
   conducted. The result of the KEGG enrichment analysis showed the
   involvement of laminarin’s targets in cancer, viral infection, cell
   death, and immune responses ([84]Figure 3A). More importantly, many
   cell signaling pathways such as Toll-like receptor signaling pathway,
   VEGF signaling pathway, p53 signaling pathway, PI3K-Akt signaling
   pathway, TNF signaling pathway, AMPK signaling pathway, NOD-like
   receptor signaling pathway, Rap1 signaling pathway, FoxO signaling
   pathway, mTOR signaling pathway, Hippo signaling pathway, JAK-STAT
   signaling pathway, and MAPK signaling pathway related to
   carcinogenicity and cell proliferation were highlighted in our result
   ([85]Figure 3B). In the Reactome pathway analysis, our result
   highlighted the contribution of laminarin to cell apoptosis through the
   regulation of TRIF-mediated programmed cell death and caspase
   activation via death receptors and extrinsic apoptotic signaling
   pathway ([86]Figure 3C). Taken together, our results suggested that the
   laminarin targets played important roles in carcinogenicity and cell
   apoptosis.

FIGURE 3.

   [87]FIGURE 3
   [88]Open in a new tab

   Laminarin’s targets involved in pathways related to cancer and cell
   apoptosis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment
   analysis highlighted the involvement of laminarin’s targets in (A)
   cancer, viral infection, cell death, and immune responses; and (B) cell
   signaling pathways related to cancer development. (C) Reactome pathway
   analysis highlighted the importance of laminarin in cell apoptosis. The
   size of a bubble represents the number of genes, and the color of the
   bubble represents the significance of the pathways.

Direct binding of laminarin to its target proteins, VEGFA, TLR4, CASP8, and
PIK3R1

   Before conducting the molecular docking analysis, laminarin’s targets
   were prioritized using the Cytoscape_v3.8.2 tool. Four core targets of
   laminarin (VEGFA, TLR4, CASP8, and PIK3R1) were obtained under the
   setting with a medium of freedom of 2.5 and the highest freedom of 5
   ([89]Figure 4A). The protein structures of VEGFA (ID: 4KZN) ([90]Shaik
   et al., 2020), TLR4 (ID: 5V3Q) ([91]Menon et al., 2017), CASP8 (ID:
   2VUW) ([92]Eswaran et al., 2009), and PIK3R1 (ID: 4JPS) ([93]Furet et
   al., 2013) were obtained from the PDB database. Then, these protein
   structures were subjected to molecular docking analysis with laminarin
   using the AutoDock Vina program, and the results were displayed using
   PyMOL (version 2.3). Our results showed the possible direct binding of
   laminarin to these four target proteins, which was reflected by the
   negative value of the binding affinity ([94]Figure 4). We found the
   formation of hydrogen bonds between laminarin with LYS-48 (3.2Å),
   HIS-86 (3.2 Å), GLN-87 (3.3Å), GLY-88 (2.2Å), and GLN-89 (3.4 Å) of
   VEGFA (ID: 4KZN) ([95]Figure 4A). The binding affinity was
   −4.2 kcal/mol. For the TLR4 (ID: 5V3Q), its amino acid residues LYS-59
   (3.1 Å), LYS-57 (2.8 Å), TYR-229 (3.0Å), PHE-31 (2.3 Å), ARG-183
   (3.3 Å), and TYP-222 (3.0Å) formed hydrogen bonds with laminarin, and
   the binding affinity was −6.2 kcal/mol ([96]Figure 4B). Similar
   bindings were observed between laminarin and CASP8 (ID: 2VUW) through
   the amino acid residues of ASP-687 (2.7 Å), GLU-492 (2.2 Å), GLN-614
   (2.7 Å), LYS-511 (2.5Å), ILE-490 (2.4 Å), and ASP-611 (2.2 Å) with
   −8.4 kcal/mol ([97]Figure 4C). In addition, laminarin formed hydrogen
   bonds with amino acid residues, ASP-933 (2.3Å), SER-774 (2.9 Å),
   ARG-770 (3.3Å), SER-854 (3.0Å), and VAL-851 (2.8 Å) of PIK3R1 (ID:
   4JPS) ([98]Figure 4D). The binding affinity was −6.9 kcal/mol. Taken
   together, our data suggested that laminarin could directly bind to its
   target proteins.

FIGURE 4.

   [99]FIGURE 4
   [100]Open in a new tab

   Direct binding of laminarin with its target proteins. Molecular docking
   analysis showed the hydrogen bond formation between laminarin with the
   amino residues of (A) VEGFA (ID: 4KZN), (B) TLR4 (ID: 5V3Q), (C) CASP8
   (ID: 2VUW), and (D) PIK3R1 (ID: 4JPS).

Discussion

   This study aimed to investigate the possible use of laminarin for
   treating cervical carcinoma and COVID-19 by targeting autophagy
   ([101]Figure 5). By searching different databases, we identified a
   cluster of common associated genes of laminarin, cervical carcinoma,
   COVID-19, and autophagy. The following genes were considered the
   possible targets of laminarin against cervical carcinoma and COVID-19
   via autophagy: CASP8, CFTR, DNMT1, HPSE, KCNH2, PIK3CA, PIK3R1,
   SERPINE1, TLR4, and VEGFA. Bioinformatic analysis, including network
   pharmacology, GO enrichment analysis, and molecular docking, was
   further used to characterize the biological functions and the
   underlying mechanisms controlled by these genes. The result of GO
   highlighted the involvement of these genes in cell death and immune
   responses, suggesting that laminarin could mediate autophagic cell
   death. Autophagy plays a dual role as a proviral and an antiviral
   factor during virus replication and is considered a target for
   developing an effective anti-SARS-CoV-2 treatment ([102]Liang et al.,
   2021). Some reports demonstrated the close association between
   autophagy and SARS-CoV-2. A kinase phosphorylation study showed that
   SARS-CoV-2 regulated various signaling pathways, including autophagy
   ([103]Chatterjee and Thakur, 2022). A genome-wide CRISPR/Cas-9 knockout
   (KO) screening suggested that autophagy-related genes were required for
   SARS-CoV-2 replication ([104]Kratzel et al., 2021). More importantly,
   studies found that induction of autophagy is essential for the
   infection and replication of SARS-CoV-2 through the regulation of acute
   inflammatory responses ([105]Zhang X. et al., 2022). On the contrary,
   autophagy also played an important role in cervical cancer. It was
   reported that autophagy controlled the epithelial-to-mesenchymal
   transition and metastasis of cervical cancer through the regulation of
   the NOTCH1 intracellular domain ([106]Zada et al., 2022). In addition,
   autophagy was crucial for the sensitivity of cervical cancer cells to
   cisplatin chemotherapy ([107]Huang et al., 2021). The application of
   laminarin to therefore target autophagy could be an alternative
   strategy for patients with COVID-19 and cervical carcinoma.

FIGURE 5.

   [108]FIGURE 5
   [109]Open in a new tab

   Workflow of the study. The pharmaceutic targets and their function
   roles were identified using network pharmacology, gene ontology
   enrichment analysis, and molecular docking.

   In the pathway analysis, we found that laminarin’s targets played a
   role in many cell signaling pathways related to COVID-19 and cervical
   carcinoma. The VEGF signaling pathway plays a vital role in the
   pathologies of COVID-19-associated pulmonary edema, sepsis, and acute
   lung injury ([110]Wang et al., 2021). A clinical comparison study among
   the COVID-19 patient categories found a significantly lower level of
   VEGF in the mildly symptomatic patients ([111]Tripathy et al., 2021).
   In addition, VEGF blockade and antagonist were reported to be helpful
   in the treatment of COVID-19 ([112]Sahebnasagh et al., 2021;
   [113]Lampropoulou et al., 2022). In cervical cancer, VEGF signaling
   regulated several oncogenic signaling pathways and cancer-regulated
   angiogenesis ([114]Prasad et al., 2022). Functionally, VEGF controlled
   proliferation and apoptosis in cervical cancer. ([115]Wu X. et al.,
   2021). Hence, VEGF was considered a therapeutic target for recurrent
   and metastatic cervical cancer ([116]Chuai et al., 2021). The VEGF
   inhibitor bevacizumab was reportedly used in advanced metastatic
   cervical cancer ([117]Singh et al., 2022). Besides VEGF signaling, we
   also found the regulation of the p53 signaling pathway by laminarin’s
   targets. This pathway was reported to be associated with COVID-19
   infection. A clinical study demonstrated that peripheral blood
   mononuclear cells of COVID-19 patients had a higher p53 expression,
   which positively correlated with the level of inflammatory cytokines
   ([118]Bordoni et al., 2021). In addition, p53 signaling is related to
   immune and apoptosis-related functions of SARS-CoV-2 infected cells
   ([119]Rahaman et al., 2021) because SARS-CoV-2 could favor tumor growth
   by inducing MDM2-mediated p53 downregulation ([120]Tanda et al., 2022)
   and target mTOR and RPS6KB1 to inhibit viral replication in the human
   respiratory tract and lung cells ([121]Ramaiah, 2020). Furthermore,
   single-nucleotide polymorphism of p53 played a role in the innate
   immune response and restricted the SARS-CoV-2-mediated mortality rate
   ([122]Lodhi et al., 2021). By targeting these signaling pathways,
   laminarin could be a potential compound to treat patients with cervical
   cancer and COVID-19.

   For the latter part, we conducted a molecular docking study to
   investigate the possible direct binding of laminarin to its core target
   proteins. Molecular docking is a key tool commonly used to predict the
   predominant binding of a ligand with a protein of known
   three-dimensional structure ([123]Morris and Lim-Wilby, 2008). Our
   result showed that laminarin had a good binding affinity to VEGFA,
   TLR4, CASP8, and PIK3R1. Vascular endothelial growth factor A (VEGFA)
   is involved in the VEGF signaling pathway, and we have discussed its
   roles in COVID-19 and cervical cancer above. Toll-like receptor 4
   (TLR4) played an important role in the generation of an antiviral state
   after being infected with pathogenic viruses ([124]Boodhoo et al.,
   2022). A study of the human pluripotent stem cell–based model of
   SARS-CoV-2 infection demonstrated the involvement of TLR4 in the
   inflammatory activation of vascular endothelial cells ([125]Ma et al.,
   2022), and TLR4 was reported to contribute to anti-inflammatory effects
   and SARS-CoV-2 infection in vitro ([126]Numata and Voelker, 2022).
   Therefore, targeting the TLR4 inflammatory pathway could serve as a
   potential strategy in reducing inflammatory lung injury ([127]Bermudez
   et al., 2022). Caspase 8 (CASP8) is a key regulator of extrinsic
   apoptosis and necroptosis pathways ([128]Tummers and Green, 2017),
   which is associated with COVID-19 infection. A study of 101 plasma
   proteins of hospitalized COVID-19 patients demonstrated a positive
   correlation between inflammatory markers and CASP8 ([129]Haljasmägi et
   al., 2020). More importantly, SARS-CoV-2 infection activated CASP8 to
   trigger cell apoptosis and inflammatory cytokine processing in the lung
   epithelial cells ([130]Li et al., 2020), suggesting CASP8 to be a
   target to relieve the severity of SARS-CoV-2 infection.

   In conclusion, our data, for the first time, provide evidence that
   laminarin could be an alternative treatment for patients with cervical
   cancer and COVID-19 by targeting autophagy. However, as our findings
   were drawn from network pharmacology, further clinical verification and
   molecular functional characterization are needed to warrant the present
   findings from bioinformatic analysis before clinical use of laminarin.

Data availability statement

   The original contributions presented in the study are included in the
   article/Supplementary Material. Further inquiries can be directed to
   the corresponding authors.

Author contributions

   WH and RL conceived and designed the study. JL, YC, LN, and XL
   performed the data analysis and data interpretation. JL, YC, LN, and XL
   conducted bioinformatics and statistical analyses. WH and RL prepared
   the manuscript.

Conflict of interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations or those of the publisher, the editors, and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

References