Combination of the extract from Phellinus linteus with Smilax spp. attenuates colorectal and prostate cancers

Technical Summary: Combination of Phellinus linteus with Smilax spp. Attenuates Colorectal and Prostate Cancers

Problem Statement
Despite the availability of conventional cancer treatments (surgery, chemotherapy, hormonal therapy), mortality rates and treatment side effects remain high. There is a growing interest in natural compounds as adjuvant therapies to improve survival and reduce toxicity. While traditional Thai herbal remedies, specifically Phellinus linteus (PL, a medicinal mushroom) and Smilax spp. (specifically Smilax corbularia and Smilax glabra, collectively denoted as PSS when combined with PL), have been used historically for cancer treatment, their specific mechanisms and efficacy against colorectal and prostate cancers require rigorous scientific validation. Previous studies on these herbs were limited to breast cancer or relied heavily on in vitro co-incubation without fully exploring immune modulation or in vivo translational potential.

Methodology
The study employed a multi-faceted approach combining in vitro cell culture, molecular analysis, immune cell modulation assays, and an in vivo xenograft model.

• Cell Lines: The study utilized three colorectal cancer cell lines (HCT116, SW620, HT-29), one prostate cancer cell line (PC3), and a non-cancerous kidney cell line (HEK 293) for toxicity and mechanism studies. Macrophages were derived from THP-1 monocytoid cells.
• Extract Preparation: A mixture (PSS) was prepared by extracting PL fruiting bodies and Smilax rhizomes in a 3:1:1 ratio using distilled water. Pure PL extract was also prepared.
• Cytotoxicity and Cell Cycle: Cytotoxicity was assessed via MTT assay to determine IC50 values. Cell cycle distribution was analyzed using flow cytometry with propidium iodide (PI) staining. Apoptosis was evaluated using Annexin V/PI staining.
• Gene Expression: RT-qPCR was performed to analyze the expression of proliferation-associated genes: MDM2, KRAS, and MKI67.
• Immune Modulation (TAMs): Tumor-associated macrophages (TAMs) were induced using HCT116 conditioned medium. The extracts' effects on TAM polarization (M1 vs. M2), cytokine secretion (TNF-α, IL-6, IL-10), and tumoricidal activity (measured via CFSE-labeled cancer cell co-incubation) were evaluated.
• In Vivo Model: A subcutaneous xenograft model was established in male nude BALB/c mice using HCT116 cells. Mice received intralesional injections of PL, PSS, or saline starting two weeks post-tumor inoculation. Tumor volume and body weight were monitored over 30 days.

Key Results

• Cytotoxicity: Both PL and PSS exhibited dose-dependent cytotoxicity against colorectal and prostate cancer cell lines. PSS demonstrated a stronger anticancer impact (lower IC50 values) than PL alone. PSS was most toxic to SW620 cells and least toxic to HEK 293 cells. Cisplatin remained the most potent agent overall.
• Cell Cycle and Apoptosis: PSS effectively induced G2 cell cycle arrest in all colorectal cell lines but did not arrest PC3 cells. PL induced G2 arrest only in HT-29 and SW620 cells. Both extracts induced low rates of apoptosis (<5%) compared to cisplatin.
• Gene Expression: Both extracts reduced MDM2 expression across all cancer cell types. MKI67 expression was reduced in HCT116, HT-29, and PC3 by both extracts. KRAS expression showed variable responses: it was downregulated in HT-29 and PC3 but upregulated in HCT116 and unchanged in SW620.
• Immune Modulation: Cancer-conditioned medium induced M2-like polarization in macrophages (upregulation of Fizz-1, TGF-β, Arg-1, IL-6, IL-10; downregulation of IL-1β, iNOS, TNF-α). Both PL and PSS reversed this trend by upregulating M1 markers (IL-1β, iNOS) and downregulating M2 markers. However, only PSS significantly reduced Arg-1 expression and demonstrated a tumoricidal effect in co-incubation assays with CFSE-stained cancer cells.
• In Vivo Efficacy: In the mouse xenograft model, administration of PSS significantly attenuated tumor volume in HCT116-injected mice. PL administration did not show a significant reduction in tumor volume, likely due to solubility limitations preventing the use of effective concentrations in vivo.

Key Contributions

1. Comparative Efficacy: The study establishes that the combination of Phellinus linteus with Smilax spp. (PSS) is more effective than Phellinus linteus alone in attenuating colorectal and prostate cancer cell proliferation in vitro and tumor growth in vivo.
2. Mechanistic Insight: The research identifies that the anticancer mechanism of PSS involves direct cytotoxicity (G2 arrest) and the modulation of the tumor microenvironment, specifically by shifting macrophage polarization from the pro-tumorigenic M2 phenotype to the anti-tumorigenic M1 phenotype.
3. Gene-Phenotype Correlation: The study highlights that the cytotoxicity of PSS does not strictly correlate with the downregulation of specific genes like MDM2 or KRAS across all cell lines, suggesting a multi-target mechanism that may be independent of specific p53 or KRAS mutation statuses.
4. Translational Proof-of-Concept: The study provides the first in vivo evidence (mouse model) that PSS can inhibit colon malignancy growth, validating its potential as a therapeutic candidate.

Significance and Claims
The authors claim that the PSS mixture holds potential clinical value as a cancer therapy, particularly for colorectal and prostate cancers. The study concludes that PSS successfully inhibits cancer cell proliferation through direct cytotoxicity and TAM modulation. However, the authors maintain a modest tone regarding immediate clinical application. They explicitly state that:

• The findings are a "proof of concept."
• Further investigation into underlying mechanisms (beyond the three genes tested) and pharmacokinetics is required.
• The current in vivo concentrations were limited by solubility, and higher concentrations or different administration routes (oral/IV) would be necessary for clinical translation.
• The extracts are not yet ready for use as herbal oral therapy or supplements without further research.
• The study acknowledges limitations, including the use of immortalized cell lines (HEK 293, THP-1) rather than primary normal cells, and the lack of combination therapy testing with standard chemotherapy.

The paper emphasizes that while PSS shows promise, its efficacy is not uniform across all cancer types or genetic backgrounds, and its application requires careful consideration of dosage and patient-specific factors.