Traditional Chinese medicine Kuan-Sin-Yin decoction inhibits cell mobility via downregulation of CCL2, CEACAM1 and PIK3R3 in hepatocellular carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Kuan-Sin-Yin (KSY) is a traditional Chinese medical decoction, designed based on the classic Si-Jun-Zi-Tang decoction and used clinically to improve the synergic effects of energy promotion, liver function and cancer related symptom and quality of life. However, the anti-hepatocellular carcinoma (HCC) function of KSY is unclear. AIM OF THE STUDY: This study aimed to investigate the anti-mobility activity of KSY on HCC cells and elucidate its molecular mechanism. MATERIALS AND METHODS: Two malignancy hepatocellular carcinoma cells, Mahlavu and SK-Hep-1, were used for the test of cell proliferation via alarm blue assay. The wound healing and Transwell assays were used to determine the anti-mobility activity of KSY in HCC cells. Cell morphology was analyzed via confocal microscopy. The genomic profile of KSY-treated HCC cells was analyzed by microarray. The potential signaling pathways and bio-functions of KSY-mediated genes were analyzed by ingenuity pathway analysis (IPA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the messenger RNA (mRNA) level of indicated gene. RESULTS: KSY did not affect cell viability of HCC cells but significantly inhibited cell migration and invasion in those HCC Mahlavu and SK-Hep-1 cells. In parallel, KSY induced changes in morphology of HCC cells via re-modulating actin cytoskeleton. KSY upregulated 1270 genes but reduced 1534 genes in Mahlavu cells. KSY regulated various gene networks which controlled cell migration, invasion and movement. Specifically, KSY reduced expression of chemokine (C-C motif) ligand 2 (CCL2), which is correlated to cell mobility, and concomitantly downregulated mRNA levels of phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) and CEA cell adhesion molecule 1 (CEACAM1). CONCLUSION: These findings indicated that regulation of CCL2-mediated PIK3R3 and CEACAM1 may be involved in KSY inhibited cell mobility. Moreover, KSY may be a potential a Chinese decoction for reducing cell mobility.

GMI, a protein from Ganoderma microsporum, induces ACE2 degradation to alleviate infection of SARS-CoV-2 Spike-pseudotyped virus.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) induces a global serious pandemic and is responsible for over 4 million human deaths. Currently, although various vaccines have been developed, humans can still get SARS-CoV-2 infection after being vaccinated. Therefore, the blocking of SARS-CoV-2 infection may be potential therapeutic strategies. Ganoderma microsporum immunomodulatory protein (GMI), a small fungal protein, is cloned from Ganoderma microsporum. It exhibits anti-cancer and immunomodulatory functions. Currently, it is still unclear whether GMI involves in interfering with viral infection. PURPOSE: This study aimed to examine the potential functions and mechanisms of GMI on inhibiting SARS-CoV-2 pseudovirus infection. METHODS: The effects of GMI were examined in vitro on ACE2 overexpressing HEK293T (HEK293T/ACE2) cells exposed to SARS-CoV-2 Spike lentiviral pseudovirus encoding a green fluorescent protein (GFP) gene. The infection efficacy was determined using fluorescence microscopy and flow cytometry. The protein level of ACE2 was verified by Western blot. The effects of GMI on cell viability of HEK293T/ACE2 and lung epithelial WI38-2RA cells were determined by MTT assay. Mice received GMI via nebulizer. RESULTS: GMI did not affect the cell viability of HEK293T/ACE2, WI38-2RA and macrophages. Functional studies showed that GMI inhibited GFP expressing SARS-CoV-2 pseudovirus from infecting HEK293T/ACE2 cells. GMI slightly interfered the interaction between ACE2 and Spike protein. GMI interacted with S2 domain of Spike protein. Specifically, GMI dramatically reduced ACE2 expression in HEK293T/ACE2 and WI38-2RA cells. Mechanistically, GMI induced ACE2 degradation via activating protein degradation system, including proteasome and lysosome. Abolishing proteasome and lysosome by MG132 and bafilomycin A1, respectively, rescued GMI-reduced ACE2 levels. In addition, GMI triggered dynamin and lipid raft-mediated ACE2 endocytosis. ACE2 levels were downregulated in the lung tissue after the mice inhaling GMI. CONCLUSIONS: GMI prevents SARS-CoV-2 pseudovirus infection via induction of ACE2 degradation in host cells. Our findings suggest that GMI will be a potential prevention agent to alleviate SARS-CoV-2 infection.

Functional proteomic analysis reveals that fungal immunomodulatory protein reduced expressions of heat shock proteins correlates to apoptosis in lung cancer cells.

BACKGROUND: Ling Zhi-8 (LZ-8) and GMI are two fungal immunomodulatory proteins (FIPs) with a similar structure and amino acid sequence and are respectively obtained from the medicinal mushroom Ganoderma lucidum and Ganoderma microsporum. They present the anti-cancer progression and metastasis. We previously demonstrated that LZ-8 reduces the tumor progression in lung cancer LLC1 cell-bearing mouse. However, it is unclear whether these FIPs induce changes in the protein expression profile in cancer cells and the mechanism for such a process is not defined. PURPOSE: This study determines the changes in the proteomic profile for tumor lesions of LLC1 cell-bearing mouse received with LZ-8 and the potential mechanism for FIPs in anti-lung cancer cells. METHODS: The proteomic profile of tumor lesions was determined using two-dimensional electrophoresis and a LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). The biological processes and the signaling pathway enrichment analysis were performed using Ingenuity Pathway Analysis (IPA). The differentially expressed proteins were verified by Western blot. Cell viability was determined by MTT assay. Cell morphology was characterized using electron microscopy. Migration was detected using the Transwell assay. The apoptotic response was determined using Western blot and flow cytometry. RESULTS: Obtained results showed that 21 proteins in the tumor lesions exhibited differential (2-fold change, p < 0.05) expression between PBS and LZ-8 treatment groups. LZ-8-induced changes in the proteomic profile that may relate to protein degradation pathways. Specifically, three heat shock proteins (HSPs), HSP60, 70 and 90, were significantly downregulated in tumor lesions of LLC1-bearing mouse received with LZ-8. Both LZ-8 and GMI reduced the protein levels for these HSPs in lung cancer cells. Functional studies showed that they inhibited cell migration but effectively induced apoptotic response in LLC1 cells in vitro. In addition, the inhibitors of HSP60 and HSP70 effectively inhibited cell migration and decreased cell viability of LLC1 cells. CONCLUSIONS: LZ-8 induced changes in the proteomic profile of tumor lesions which may regulate the HSPs-related cell viability. Moreover, inhibition of HSPs may be related to the anti-lung cancer activity.