RNA-seq transcriptome and pathway analysis of the medicinal mushroom Lignosus tigris (Polyporaceae) offer insights into its bioactive compounds with anticancer and antioxidant potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal mushrooms belonging to the Lignosus spp., colloquially known as Tiger Milk mushrooms (TMMs), are used as traditional medicine by communities across various regions of China and Southeast Asia to enhance immunity and to treat various diseases. At present, three Lignosus species have been identified in Malaysia: L. rhinocerus, L. tigris, and L. cameronensis. Similarities in their macroscopic morphologies and the nearly indistinguishable appearance of their sclerotia often lead to interchangeability between them. Hence, substantiation of their traditional applications via identification of their individual bioactive properties is imperative in ensuring that they are safe for consumption. L. tigris was first identified in 2013. Thus far, studies on L. tigris cultivar sclerotia (Ligno TG-K) have shown that it possesses significant antioxidant activities and has greater antiproliferative action against selected cancer cells in vitro compared to its sister species, L. rhinocerus TM02®. Our previous genomics study also revealed significant genetic dissimilarities between them. Further omics investigations on Ligno TG-K hold immense potential in facilitating the identification of its bioactive compounds and their associated bioactivities. AIM OF STUDY: The overall aim of this study was to investigate the gene expression profile of Ligno TG-K via de novo RNA-seq and pathway analysis. We also aimed to identify highly expressed genes encoding compounds that contribute to its cytotoxic and antioxidant properties, as well as perform a comparative transcriptomics analysis between Ligno TG-K and its sister species, L. rhinocerus TM02®. MATERIALS AND METHODS: Total RNA from fresh 3-month-old cultivated L. tigris sclerotia (Ligno TG-K) was extracted and analyzed via de novo RNA sequencing. Expressed genes were analyzed using InterPro and NCBI-Nr databases for domain identification and homology search. Functional categorization based on gene functions and pathways was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Genes (COG) databases. Selected genes were subsequently subjected to phylogenetic analysis. RESULTS: Our transcriptomics analysis of Ligno TG-K revealed that 68.06% of its genes are expressed in the sclerotium; 80.38% of these were coding transcripts. Our analysis identified highly expressed transcripts encoding proteins with prospective medicinal properties. These included serine proteases (FPKM = 7356.68), deoxyribonucleases (FPKM = 3777.98), lectins (FPKM = 3690.87), and fungal immunomodulatory proteins (FPKM = 2337.84), all of which have known associations with anticancer activities. Transcripts linked to proteins with antioxidant activities, such as superoxide dismutase (FPKM = 1161.69) and catalase (FPKM = 1905.83), were also highly expressed. Results of our sequence alignments revealed that these genes and their orthologs can be found in other mushrooms. They exhibit significant sequence similarities, suggesting possible parallels in their anticancer and antioxidant bioactivities. CONCLUSION: This study is the first to provide a reference transcriptome profile of genes expressed in the sclerotia of L. tigris. The current study also presents distinct COG profiles of highly expressed genes in Ligno TG-K and L. rhinocerus TM02®, highlighting that any distinctions uncovered may be attributed to their interspecies variations and inherent characteristics that are unique to each species. Our findings suggest that Ligno TG-K contains bioactive compounds with prospective medicinal properties that warrant further investigations. CLASSIFICATION: Systems biology and omics.

Evaluation of the proteomic landscape of HPV E7­induced alterations in human keratinocytes reveal therapeutically relevant pathways for cervical cancer.

The lack of specific and accurate therapeutic targets poses a challenge in the treatment of cervical cancer (CC). Global proteomics has the potential to characterize the underlying and intricate molecular mechanisms that drive the identification of therapeutic candidates for CC in an unbiased manner. The present study assessed human papillomavirus (HPV)­induced proteomic alterations to identify key cancer hallmark pathways and protein­protein interaction (PPI) networks, which offered the opportunity to evaluate the possibility of using these for targeted therapy in CC. Comparative proteomic profiling of HPV­transfected (HPV16/18 E7), HPV­transformed (CaSki and HeLa) and normal human keratinocyte (HaCaT) cells was performed using the liquid chromatography­tandem mass spectrometry (LC­MS/MS) technique. Both label­free quantification and differential expression analysis were performed to assess differentially regulated proteins in HPV­transformed and ­transfected cells. The present study demonstrated that protein expression was upregulated in HPV­transfected cells compared with in HPV­transformed cells. This was probably due to the ectopic expression of E7 protein in the former cell type, in contrast to its constitutive expression in the latter cell type. Subsequent pathway visualization and network construction demonstrated that the upregulated proteins in HPV16/18 E7­transfected cells were predominantly associated with a diverse array of cancer hallmarks, including the mTORC1 signaling pathway, MYC targets V1, hypoxia and glycolysis. Among the various proteins present in the cancer hallmark enrichment pathways, phosphoglycerate kinase 1 (PGK1) was present across all pathways. Therefore, PGK1 may be considered as a potential biomarker. PPI analysis demonstrated a direct interaction between p130 and polyubiquitin B, which may lead to the degradation of p130 via the ubiquitin­proteasome proteolytic pathway. In summary, elucidation of the key signaling pathways in HPV16/18­transfected and ­transformed cells may aid in the design of novel therapeutic strategies for clinical application such as targeted therapy and immunotherapy against cervical cancer.

In vivo anti-tumor activity of Lignosus rhinocerus TM02® using a MCF7-xenograft NCr nude mice model.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerus (Cooke) Ryvarden (also known as Tiger Milk mushroom, TMM), is a basidiomycete belonging to the Polyporaceae family. It has been documented to be used by traditional Chinese physicians and indigenous people in Southeast Asia to treat a variety of illnesses, such as gastritis, arthritis, and respiratory conditions, as well as to restore patients' physical well-being. TMM has also been used in folk medicine to treat cancer. For example, people from the indigenous Kensiu tribe of northeast Kedah (Malaysia) apply shredded TMM sclerotium mixed with water directly onto breast skin to treat breast cancer, while Chinese practitioners from Hong Kong, China prescribe TMM sclerotium as a treatment for liver cancer. L. rhinocerus has previously been demonstrated to possess selective anti-proliferative properties in vitro, however pre-clinical in vivo research has not yet been conducted. AIM OF STUDY: This study aimed to examine the anti-tumor activities of L. rhinocerus TM02®, using two different sample preparations [cold water extract (CWE) and fraction] via various routes of administration (oral and intraperitoneal) on an MCF7-xenograft nude mouse model. This study also investigated the inhibitory effect of TM02® CWE and its fractions against COX-2 in vitro using LPS-induced RAW264.7 macrophages, on the basis of the relationship between COX-2 and metastasis, apoptosis resistance, as well as the proliferation of cancer cells. MATERIALS AND METHODS: The first preparation, L. rhinocerus TM02® sclerotium powder (TSP) was dissolved in cold water to obtain the cold water extract (CWE). It was further fractionated based on its molecular weight to obtain the high (HMW), medium (MMW) and low (LMW) molecular weight fractions. The second preparation, known as the TM02® rhinoprolycan fraction (TRF), was obtained by combining the HMW and MMW fractions. TSP was given orally to mimic the daily consumption of a supplement; TRF was administered intraperitoneally to mimic typical tumorous cancer treatment with a rapid and more thorough absorption through the peritoneal cavity. Another experiment was conducted to examine changes in COX-2 activity in LPS-induced RAW264.7 macrophages after a 1-h pre-treatment with CWE, HMW, and MMW. RESULTS: Our results revealed that intraperitoneal TRF-injection (90 µg/g BW) for 20 days reduced initial tumor volume by ∼64.3% (n = 5). The percentage of apoptotic cells was marginally higher in TRF-treated mice vs. control, suggesting that induction of apoptosis as one of the factors that led to tumor shrinkage. TSP (500 µg/g BW) oral treatment (n = 5) for 63 days (inclusive of pre-treatment prior to tumor inoculation) effectively inhibited tumor growth. Four of the five tumors totally regressed, demonstrating the effectiveness of TSP ingestion in suppressing tumor growth. Although no significant changes were found in mouse serum cytokines (TNF-α, IL-5, IL-6 and CCL2), some increasing and decreasing trends were observed. This may suggest the immunomodulatory potential of these treatments that can directly or indirectly affect tumor growth. Pre-treatment with CWE, HMW and MMW significantly reduced COX-2 activity in RAW264.7 macrophages upon 24 h LPS-stimulation, suggesting the potential of L. rhinocerus TM02® extract and fractions in regulating M1/M2 polarization. CONCLUSION: Based on the findings of our investigation, both the rhinoprolycan fraction and crude sclerotial powder from L. rhinocerus TM02® demonstrated tumor suppressive effects, indicating that they contain substances with strong anticancer potential. The antitumor effects of L. rhinocerus TM02® in our study highlights the potential for further explorations into its mechanism of action and future development as a prophylactic or adjunct therapeutic against tumorous cancer.

In silico analysis and characterization of medicinal mushroom cystathionine beta-synthase as an angiotensin converting enzyme (ACE) inhibitory protein.

Angiotensin-converting enzyme (ACE) regulates blood pressure and has been implicated in several conditions including lung injury, fibrosis and Alzheimer's disease. Medicinal mushroom Ganordema lucidum (Reishi) cystathionine beta-synthase (GlCBS) was previously reported to possess ACE inhibitory activities. However, the inhibitory mechanism of CBS protein remains unreported. Therefore, this study integrates in silico sequencing, structural and functional based-analysis, protein modelling, molecular docking and binding affinity calculation to elucidate the inhibitory mechanism of GlCBS and Lignosus rhinocerus (Tiger milk mushroom) CBS protein (LrCBS) towards ACE. In silico analysis indicates that CBSs from both mushrooms share high similarities in terms of physical properties, structural properties and domain distribution. Protein-protein docking analysis revealed that both GlCBS and LrCBS potentially modulate the C-terminal domain of ACE (C-ACE) activity via regulation of chloride activation and/or prevention of substrate entry. GICBS and LrCBS were also shown to interact with ACE at the same region that presumably inhibits the function of ACE.

Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells.

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.