KMP01D Demonstrates Beneficial Anti-inflammatory Effects on Immune Cells: An ex vivo Preclinical Study of Patients With Colorectal Cancer

Abstract

Background: Colorectal cancer (CRC) is frequently associated with dysbiosis of the gut microbiome which, together with a compromised gut barrier, can result in perioperative endotoxin leakage into the circulation. Constant local and systemic inflammatory activity is suggested to facilitate metastases formation. Previous studies have pointed to the capacity of a colostrum preparation to neutralize endotoxins within the gastrointestinal tract which could ameliorate associated inflammatory responses and tumor recurrence in affected patients. This study aimed to examine the effects of the colostrum preparation, KMP01D, on the inflammatory activity of patient-derived immune cells. Methods: The effects of KMP01D on pro-/anti-inflammatory cytokine responses and apoptosis were examined ex vivo using immune cells from CRC patients (stages I-IV, n = 48). The expression of CD14, CD68, Toll-like receptor (TLR)4, and insulin-like growth factor (IGF)-1 was also analyzed. Results: KMP01D increased interleukin (IL)-10 and IL-13 anti-inflammatory cytokine expression in patient-derived peripheral blood mononuclear cells (PBMCs). Interestingly, KMP01D also decreased the secretion of IL-1β, IL-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-12 inflammatory cytokines, and IGF-1 in these cells. Moreover, CD14 and TLR4 expression involved in endotoxin signaling was downregulated in PBMCs and tumor-derived cells. Apoptosis of immune cells and tumor-derived cells was likewise enhanced with KMP01D. Addition of vitamin D3 as a cofactor demonstrated enhanced anti-inflammatory effects. Conclusions: KMP01D demonstrated beneficial ex vivo effects on inflammatory cytokine responses in PBMCs and enhanced apoptosis of immune cells from CRC patients. In line with previous clinical trials, we present new evidence endorsing KMP01D as a treatment strategy to regulate stage-dependent local and systemic inflammation in CRC patients.

Keywords: KMP01D; colorectal cancer; immune regulator; inflammation; vitamin D3.

Figure 1

Schematic overview of the proposed neutralization potential of polyvalent immunoglobulins (PVIGs) found in KMP01D. Orally ingested colostral PVIGs of bovine origin pass through the gut lumen where they bind and thereby neutralize endotoxin [lipopolysaccharide (LPS)] or opsonize bacteria (such as Escherichia coli). As unmodified and therefore foreign proteins, processing of orally administered PVIGs proceeds either by passive excretion or internalization of receptor-bound PVIGs by monocytes/macrophages following transcytosis or leakage through the epithelial layer.

Figure 2

Comparative analysis of monocyte frequency (CD14⁺CD45⁺ cells) determined by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 12) and Union for International Cancer Control (UICC) stage I–IV colorectal cancer (CRC) patients (n = 12 for each stage). Decreased expression of CD14⁺CD45⁺ cells was observed in the PBMCs of UICC stage I–III CRC patients incubated with KMP01D and the combination of KMP01D and vitamin D3 (UICC I: p < 0.05 and p < 0.01, UICC II: p < 0.001 and p < 0.001, UICC III: p < 0.05 and p < 0.01, respectively).

Figure 3

RT-qPCR analysis showing decreased (A) CD14, (B) CD68, (C) Toll-like receptor (TLR)4, and (D) insulin-like growth factor (IGF)-1 gene expression in peripheral blood mononuclear cells (PBMCs) from Union for International Cancer Control (UICC) stage I–III colorectal cancer (CRC) patients (n = 12 for each stage) following incubation with KMP01D and the combination of KMP01D and vitamin D3 compared with untreated cells (CD14: UICC I–II: ^**p < 0.0001 and ^*p < 0.0001, UICC III: ^*p < 0.001 and ^*p < 0.0001; CD68: UICC I–II: ^**p < 0.0001 and ^*p < 0.0001, UICC III: ^*p < 0.001 and ^*p < 0.0001; TLR4: UICC I: p > 0.05 and ^*p < 0.001, UICC II: ^*p < 0.001 and ^**p < 0.001, UICC III: p > 0.05 and ^**p < 0.01; IGF-1: UICC I–II: ^*p < 0.001 and ^*p < 0.001, UICC III: ^**p = 0.02 and ^**p = 0.02, respectively). An enhanced effect was observed after incubation with the combination of KMP01D and vitamin D3.

Figure 4

Analysis for apoptosis of peripheral blood mononuclear cells (PBMCs) and tumor-derived cells from healthy volunteers (n = 12) and colorectal cancer (CRC) patients (n = 12 for each stage). Increased apoptosis of (A) CD68⁺ PBMCs [representative TUNEL assay immunofluorescence staining of Union for International Cancer Control (UICC) stage III], (B) CD68⁺ tumor-derived cells [representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay immunofluorescence staining of UICC stage III], and (C) CD14⁺CD95⁺ PBMCs [representative immunofluorescence staining of UICC stage III for expression of CD95 (i.e., Fas)] following incubation with KMP01D. An enhanced effect was observed following incubation with the combination of KMP01D and vitamin D3. All images were obtained at 200 × or 400 × magnification. TUNEL-fluorescein isothiocyanate (FITC), green; anti-CD68-cyanine 3 (Cy3), pink; CD14-FITC; CD95-Cy3.

Figure 5

RT-qPCR analysis of cell fractions from tumor tissues showing decreased (A) CD14, (B) CD68, (C) Toll-like receptor (TLR)4, and (D) insulin-like growth factor (IGF)-1 gene expression in tumor-derived cells of Union for International Cancer Control (UICC) stage I–IV patients (n = 12 for each stage) following incubation with KMP01D and the combination of KMP01D and vitamin D3 compared with untreated cells. (CD14: UICC I–III: ^*p < 0.0001 and ^*p < 0.0001; CD68: UICC I–III: ^**p < 0.0001 and ^**p < 0.0001; TLR4: UICC I–III: ^*p < 0.001 and ^*p < 0.001; IGF-1: UICC I–II: ^*p < 0.001 and ^*p < 0.001, UICC III: ^**p < 0.01 and ^**p < 0.01, respectively). An enhanced effect on decreasing CD14 and CD68 expression was observed after incubation with the combination of KMP01D and vitamin D3 (UICC I–III).