An immune-stimulating proteoglycan from the medicinal mushroom Huaier up-regulates NF-κB and MAPK signaling via Toll-like receptor 4

Target Article

Reliability Check of the Study (PICO)

Study Design and Sample Size

Detailed Results

[Physicochemical Characteristics of the Huaier-Derived TPG-1 Complex]

According to the study reported by Ailin Yang and colleagues in 2019, TPG-1, a proteoglycan purified from an aqueous extract of Huaier, was shown to possess distinct structural characteristics.

Molecular analyses demonstrated that TPG-1 had an average molecular weight of approximately 5.59 × 10^4 Da, with carbohydrates accounting for 43.93% and proteins for 41.20% of its total composition.

[In Vitro Antitumor Effects Mediated by Macrophage Activation]

Under in vitro conditions, TPG-1 itself showed only a very limited direct inhibitory effect on the proliferation of the human hepatocellular carcinoma cell lines HepG2 and SK-HEP-1.

However, when cancer cells were exposed to culture supernatants obtained from murine macrophage-like RAW264.7 cells treated with TPG-1, time-dependent cytotoxic effects against the cancer cells were observed.

In particular, a significant reduction in hepatocellular carcinoma cell viability was confirmed after 72 hours of exposure (P<0.001).

These findings suggest that soluble factors released from TPG-1-treated macrophages contribute to the indirect antitumor effects observed in vitro, rather than TPG-1 acting solely through direct cytotoxicity against tumor cells. The cytotoxic effect of the macrophage-conditioned medium increased over time.

[Induction of Nitric Oxide Production and Cytokine Expression in Macrophages]

Nitric oxide (NO) production in RAW264.7 cells was significantly increased by TPG-1 compared with the control level of 4.85 ± 0.34 μM.

No clear concentration-dependent increase in NO production was observed across the tested TPG-1 concentrations, although all TPG-1-treated groups differed significantly from the control group (all P<0.001).

In contrast, TNF-α production increased in a dose-dependent manner following stimulation with TPG-1.

In addition, TPG-1 increased IL-6 mRNA expression and secretion, as well as the mRNA and protein expression of iNOS and COX-2.

These findings demonstrate that TPG-1 stimulates multiple immune- and inflammation-related responses in RAW264.7 macrophages.

[Activation of NF-κB and MAPK Signaling Through TLR4]

RNA sequencing identified the Toll-like receptor, TNF, and NF-κB signaling pathways among the major pathways affected by TPG-1 treatment.

Immunoblotting showed that TPG-1 increased phosphorylation of components of the NF-κB pathway, including IKKα/β, IκBα, and p65. TPG-1 also increased phosphorylation of the MAPK proteins p38, ERK, and JNK.

The involvement of TLR4 was examined using TAK-242, a TLR4 inhibitor.

TAK-242 markedly attenuated the TPG-1-induced activation of both NF-κB and MAPK signaling. It also reduced the TPG-1-induced production of TNF-α, IL-6, and NO.

Consistent findings were obtained when TLR4 expression was suppressed using siRNA. Reduction of TLR4 expression attenuated the increases in TNF-α, IL-6, and NO induced by TPG-1.

These findings provide molecular evidence that TPG-1-induced macrophage activation in RAW264.7 cells is dependent, at least to a substantial extent, on TLR4 signaling and downstream activation of the NF-κB and MAPK pathways.

[In Vivo Tumor Growth Inhibition and Immune-Cell Infiltration]

In vivo experiments demonstrated that intraperitoneal administration of TPG-1 at 60 mg/kg once daily significantly inhibited the growth of HepG2 tumors in nude mice and H22 tumors in Kunming mice. Significant differences were observed by day 16 of treatment (P<0.001 or P<0.01).

In the HepG2 xenograft model, TPG-1 treatment was associated with increased serum TNF-α levels and increased infiltration of CD45-positive leukocytes and F4/80-positive macrophages into tumor tissue.

Increased immune-cell infiltration was also observed in tumors from H22-bearing mice treated with TPG-1.

These findings support the authors’ interpretation that immune potentiation contributes, at least in part, to the antitumor activity of TPG-1 in vivo.

However, the animal experiments did not establish that TLR4-dependent signaling alone was responsible for the observed inhibition of tumor growth.

[In Vivo Safety and Toxicity Assessment]

No significant body-weight loss was observed in HepG2-bearing nude mice treated with TPG-1. In H22-bearing Kunming mice, TPG-1 treatment was associated with a slight suppression of body-weight gain.

Histological examination revealed no detectable tissue toxicity in major organs, including the heart, liver, spleen, lungs, and kidneys.

In addition, hematological analyses showed no detectable toxicity affecting white blood cell, red blood cell, or platelet counts.

Overall, these findings indicate that TPG-1 was generally well tolerated under the experimental conditions used in these mouse models. However, they should not be interpreted as demonstrating comprehensive or long-term safety in other species.

Potential Applications in Veterinary Medicine 

[How to Utilize in Clinical Practice]

The mechanism demonstrated in this study is notable because TPG-1 does not appear to exert its main effect by directly attacking cancer cells. Instead, it activates the host immune system, particularly macrophages, and thereby influences the tumor microenvironment.

In routine veterinary oncology, standard treatments such as surgery and chemotherapy remain the first-line options. However, clinicians frequently encounter patients in which such treatments cannot be performed because of advanced age, concerns regarding adverse effects, or other clinical factors.

Because the molecular mechanisms of TPG-1 have been characterized to some extent, these findings provide a rationale for further investigation of TPG-1 as a potential adjunctive approach in veterinary oncology. However, the present study does not provide direct evidence of efficacy in dogs or cats.

[Comparison With Existing Treatments]

5-Fluorouracil (5-FU) was included as a positive control in the animal experiments.

Both TPG-1 and 5-FU inhibited tumor growth under the experimental conditions; however, the study was not designed to establish therapeutic equivalence between the two treatments. TPG-1 and 5-FU were not administered in combination, and therefore additive or synergistic effects cannot be concluded from this study.

Rather than supporting replacement of established anticancer treatments, these findings provide a rationale for further investigation of TPG-1 as a potential adjunctive approach.

[Study Limitations and Critical Appraisal]

The authors noted that it remains unclear whether TPG-1 is a key component responsible for the clinical effects attributed to Huaier.

Furthermore, the study consists of in vitro experiments using cell lines and in vivo experiments using mouse tumor models.

Accordingly, caution is required when extrapolating these findings directly to clinical use in dogs or cats.

Mini-Glossary for Readers

TPG-1:
An immune-stimulating proteoglycan isolated from Huaier, consisting of carbohydrates and proteins in approximately comparable proportions.

TLR4 (Toll-like receptor 4):
A receptor involved in the recognition of pathogen-associated molecular patterns. When activated, it transmits intracellular signals that induce immune responses.

NF-κB / MAPK pathways:
Major intracellular signaling pathways involved in the regulation of cellular proliferation and differentiation, as well as immune and inflammatory responses.

Link to the full article: https://doi.org/10.1074/jbc.RA118.005477