Assessing the Therapeutic Impacts of HAMLET and FOLFOX on BRAF-Mutated Colorectal Cancer: A Study of Cancer Cell Survival and Mitochondrial Dynamics In Vitro and Ex Vivo

Abstract

Background and Objectives: Colorectal cancer (CRC) is a major global health challenge. The BRAF V600E mutation, found in 8-12% of CRC patients, exacerbates this by conferring poor prognosis and resistance to therapy. Our study focuses on the efficacy of the HAMLET complex, a molecular substance derived from human breast milk, on CRC cell lines and ex vivo biopsies harboring this mutation, given its previously observed selective toxicity to cancer cells. Materials and Methods: we explored the effects of combining HAMLET with the FOLFOX chemotherapy regimen on CRC cell lines and ex vivo models. Key assessments included cell viability, apoptosis/necrosis induction, and mitochondrial function, aiming to understand the mutation-specific resistance or other cellular response mechanisms. Results: HAMLET and FOLFOX alone decreased viability in CRC explants, irrespective of the BRAF mutation status. Notably, their combination yielded a marked decrease in viability, particularly in the BRAF wild-type samples, suggesting a synergistic effect. While HAMLET showed a modest inhibitory effect on mitochondrial respiration across both mutant and wild-type samples, the response varied depending on the mutation status. Significant differences emerged in the responses of the HT-29 and WiDr cell lines to HAMLET, with WiDr cells showing greater resistance, pointing to factors beyond genetic mutations influencing drug responses. A slight synergy between HAMLET and FOLFOX was observed in WiDr cells, independent of the BRAF mutation. The bioenergetic analysis highlighted differences in mitochondrial respiration between HT-29 and WiDr cells, suggesting that bioenergetic profiles could be key in determining cellular responses to HAMLET. Conclusions: We highlight the potential of HAMLET and FOLFOX as a combined therapeutic approach in BRAF wild-type CRC, significantly reducing cancer cell viability. The varied responses in CRC cell lines, especially regarding bioenergetic and mitochondrial factors, emphasize the need for a comprehensive approach considering both genetic and metabolic aspects in CRC treatment strategies.

Keywords: BRAF mutation; HAMLET; bioactive milk compound; colorectal cancer; ex vivo treatment; mitochondrial function; precision medicine.

Chart 1

Overall flow chart design of colorectal surgeries in 2021 and 2022 and inclusion for study. IBD—inflammatory bowel disease. * We only included patients operated on Monday or Tuesday due to methodology.

Figure 1

Establishing the explant model: 1—a piece of tumor and healthy tissue is collected after operation and examination; 2—the pieces are washed three times under sterile conditions; 3—the pieces are cut into 2 mm pieces with a biopsy needle, avoiding shredding as much as possible; 4—we are left with 2 mm³ explants; 5—every single explant is placed into a well of 96-well plate; and 6—incubation at 37 °C, 95—98% humidity, and 5% CO₂ atmosphere.

Figure 2

Kaplan–Meier analysis by month, stratified by mutation status.

Figure 3

The effect of 60 μM of HAMLET and/or FOLFOX (3 mM of 5-FU + 75 μM of oxaliplatin) on CRC explant viability. (a) Explant viability 24 h after treatment; (b) explant viability 48 h after treatment. Comparing BRAF wild-type and mutant explants. Means ± SD. N ≥ 3. Dotted line—control group data (100%). *—p < 0.05 comparing with control group data.

Figure 4

Comparison of the impacts of HAMLET and control on mitochondrial functions between BRAF wild type and BRAF mutant: (a) mitochondrial non-phosphorylating (V0) respiration rate and (b) mitochondrial state 3 (VADP) respiration rate are means ± SD. *—p < 0.05 comparing non-treated control and HAMLET-treated samples.

Figure 5

Dose-dependent responses of CRC cell lines to HAMLET complex. (a) The MTT assay performed 24 h after a 6 h incubation revealed a dose-dependent response and higher WiDr cell line resistance to HAMLET than HT-29. (b) The effect of HAMLET on colony formation in different BRAF mutant CRC cell lines. Graphic representation and representative photos. (c) Apoptotic cell line population after the effect of HAMLET. (d) Necrotic cell line population after the effect of HAMLET. Control group data—dotted line. * p  <  0.05—compared with control group data. ** p  <  0.05—comparing HAMLET’s effects on WiDr and HT-29. ***—no colony formation. **** p  ≤  0.05 when comparing apoptosis and necrosis of the same sample. Means ± SD. N ≥ 3.

Figure 6

Cell responses to FOLFOX. (a) Dose–response graph of WiDr’s and HT-29′s responses to different doses of FOLFOX. (b) FOLFOX IC50 dose calculation for WiDr cell line. (c) FOLFOX IC50 dose calculation for HT-29 cell line. N = 3; means ± SD. Purple dot marks 50% viability (IC50).

Figure 7

Heatmaps and tables of Bliss synergy and antagonism calculation. (a) WiDr cell line; (b) HT-29 cell line. Heatmaps show decrease in viability when treating with varying doses of FOLFOX and/or HAMLET compared with control (100% viability). Tables show synergy or antagonism index. Higher positive number with blue color shows significant synergy, and negative number with red color shows significant antagonism (not present). *—p < 0.05.

Figure 8

Comparison of the mitochondrial and glycolytic activity of WiDr and HT-29 cells. In (a), mitochondrial oxygen consumption curves are presented as averages ± standard deviations of each measurement time point (n = 3 of 3 technical replicates). In (b), summarized mitochondrial capacity data are calculated from the curves in (a). * and **—statistically significant differences compared with WiDr; p < 0.05 and p < 0.01, respectively. In (c), glycolytic activity is monitored as pH changes simultaneously with the oxygen consumption rate. In (d), energy phenotype plots represent mitochondrial and glycolytic energy capacity distributions under normal (basal) and stressed conditions. * and **—statistically significant differences compared with basal conditions for the same cell type; p < 0.05 and p < 0.01, respectively. ^—statistically significant difference compared with the same parameter of WiDr; p < 0.05. OCR—oxygen consumption rate, ECAR—extracellular acidification rate, Oli—oligomycin, FCCP—carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, Ro—rotenone, and AA—antimycin A.

Figure 9

Effect of HAMLET on mitochondrial respiration and respiratory control index (RCI). Mitochondrial respiration rate was measured as described in “Methods”. (a) Mitochondrial non-phosphorylating (V₀) respiration rate in the presence of 1 mln/mL of cells and glutamate (5 mM) plus malate (2 mM); (b) state 3 respiration rate in the presence of ADP (1 mM; VADP); (c) mitochondrial maximal respiration rate in the presence of succinate (12 mM; Vsucc); (d) mitochondrial respiration rate in the presence of cytochrome c (32 μM; Vcyt c); and (e) mitochondrial respiratory control index (RCI) (VADP/V0)). * p  <  0.05 compared with the control group.

Figure 10

Schematic representation of the possible combined effect of HAMLET and FOLFOX components (oxaliplatin and 5-fluorouracil). The combined effect of FOLFOX component oxaliplatin and HAMLET is most likely exerted via an effect on mitochondria. HAMLET decreases cell ATP production and cell respiration, and our previous studies show that platin-based drugs reduce ATP production, respiration, and mitochondria membrane permeability (Caco-2, AGS, and T3M4 with cisplatin). The combined effect of HAMLET and platin-based drugs on mitochondria can, in some cases, have synergistic effects, leading to increased cell death via mitochondrial damage. ↑ increaced ↓ decreased.