Colorectal cancer (CRC) is one of the most common cancers worldwide and remains deadly because tumors frequently come back or spread after treatment. Despite improvements in surgery and chemotherapy, recurrence and metastasis continue to be major obstacles for patients.
An emerging area of research asks whether drugs used during surgery - especially anesthetic agents - might directly affect tumor cells beyond their primary role of putting patients to sleep. This study focused on ketamine, an NMDA receptor antagonist commonly used in anesthesia, which had already shown anti-cancer effects in gastric, lung, and breast cancer models.
The researchers wanted to know whether ketamine could stop HT-29 cells - a human colorectal adenocarcinoma cell line - from growing and push them toward programmed cell death (apoptosis). If ketamine can do double duty as both anesthetic and tumor suppressor, it could be particularly valuable during and after colorectal cancer surgery.
HT-29 colorectal cancer cells were grown in standard culture conditions and treated with ketamine at four concentrations: 0.5, 1, 2.5, and 5 micromolar (µM). Cell survival was measured using the MTT assay, a colorimetric test that measures how many cells are alive and metabolically active. All experiments were run in triplicate across three independent rounds.
To measure apoptosis (programmed cell death), the researchers used flow cytometry with Annexin V-FITC and 7-AAD staining, which can distinguish between healthy cells, early dying cells, late dying cells, and necrotic (accidentally destroyed) cells. Cells were treated with 1 µM ketamine for 24 hours before this analysis.
Gene expression changes were evaluated using a quantitative PCR (qPCR) array covering 29 genes involved in oncogenic signaling pathways like Wnt/beta-catenin and EGFR signaling. The researchers noted this was a basal cell carcinoma panel (not CRC-specific), which they acknowledged as a limitation. Finally, molecular docking simulations using two independent computational methods (AutoDock Vina and Schrödinger Glide) were run to predict how ketamine binds to three target proteins: the NMDA receptor, EGFR, and CSNK1D.
The MTT assay results were striking: ketamine reduced HT-29 cell viability progressively with each increasing dose. At 0.5 µM, viability dropped to 70%; at 1 µM it fell to 52%; at 2.5 µM to 30%; and at 5 µM only 17% of cells were still alive. The IC50 value - the concentration that kills half the cancer cells - was approximately 1.05 µM, indicating potent activity at very low concentrations.
Statistical analysis confirmed these results were highly significant (one-way ANOVA, F = 98.96, p less than 0.001). All ketamine-treated groups showed significantly reduced viability compared to untreated controls, with p less than 0.01 at the lowest dose and p less than 0.001 at higher doses. This dose-response relationship is a hallmark of genuine drug-induced cell killing.
The IC50 of roughly 1.05 µM is comparable to, or even lower than, those reported for several established anti-cancer drugs tested in colorectal cancer cell models - suggesting ketamine may exert biologically meaningful effects at concentrations that could potentially be achievable in clinical settings.
Beyond simply reducing cell counts, the researchers wanted to know how cells were dying. Flow cytometry with Annexin V/7-AAD staining showed that after 24 hours of 1 µM ketamine treatment, 23.9% of cells were in early apoptosis - a form of controlled, programmed cell death. In untreated control cells, this proportion was minimal.
Late apoptotic cells accounted for only 1.89% of the population, and necrotic cells (which die in a messy, inflammatory way) were negligible at 0.37%. This distinction matters clinically: early apoptosis is a clean, non-inflammatory form of cell death that is less likely to cause tissue damage or trigger harmful inflammatory responses in surrounding healthy tissue.
Together, the viability and apoptosis data confirm that ketamine kills colorectal cancer cells primarily by triggering the apoptotic program - the cell's own built-in self-destruction pathway - rather than by physically rupturing cells or causing random damage.
The qPCR gene expression analysis of 29 cancer-related genes revealed that three genes were significantly downregulated (fold change below 0.5) in ketamine-treated HT-29 cells: EGF (Epidermal Growth Factor, fold change 0.39), TCF7 (Transcription Factor 7, fold change 0.41), and CSNK1D (Casein Kinase 1 Delta, fold change 0.25). All three met the threshold for significant downregulation (p less than 0.05 or p less than 0.001).
EGF is a growth signal that activates the MAPK/ERK and PI3K/AKT pathways, which drive cell growth, survival, and invasiveness. Its downregulation could suppress the cancer cells' ability to invade surrounding tissue. TCF7 is a key transcription factor in the Wnt/beta-catenin pathway, which is frequently hyperactivated in colorectal cancer - its suppression would dampen a major driver of tumor growth.
CSNK1D is a serine/threonine kinase involved in stabilizing beta-catenin and coordinating multiple pro-survival pathways. Its downregulation by ketamine disrupts these coordinated signaling networks. Notably, 26 of the 29 other genes tested showed no significant change, suggesting ketamine's gene-level effects are somewhat targeted rather than broadly toxic to all gene expression.
To understand how ketamine interacts with cancer-relevant proteins at the molecular level, the researchers used two independent molecular docking methods - AutoDock Vina and Schrödinger Glide - to simulate how tightly ketamine binds to three proteins: the NMDA receptor, EGFR, and CSNK1D. Using two methods adds reliability since each has different scoring approaches.
Both methods consistently ranked the NMDA receptor as the strongest target: AutoDock Vina predicted a binding energy of -6.79 kcal/mol, while Schrödinger Glide produced -7.53 kcal/mol. By contrast, binding to EGFR and CSNK1D was substantially weaker (-6.00 vs. -4.43 kcal/mol for EGFR; -5.90 vs. -5.96 kcal/mol for CSNK1D). A lower (more negative) binding energy means stronger, more stable binding.
Structural analysis showed ketamine fits snugly inside the hydrophobic channel of the NMDA receptor pore, forming critical contacts with residues Ala644, Val640, Leu643, and a key hydrogen bond with Thr647. This is consistent with ketamine's known mechanism of physically blocking the NMDA receptor channel. The weak binding to EGFR and CSNK1D suggests their gene expression changes are likely downstream effects of NMDA blockade, not direct drug-protein interactions.
The finding that EGFR and CSNK1D genes were suppressed even though ketamine doesn't strongly bind these proteins seems puzzling at first. The explanation lies in interconnected signaling cascades: blocking the NMDA receptor reduces calcium ions entering the cell, which in turn decreases activation of CaMKII (calcium/calmodulin-dependent protein kinase II) and the oncogene c-Myc. Since c-Myc controls expression of many proliferative genes, its suppression can ripple out to affect EGF and CSNK1D expression.
Further, there is documented crosstalk between the NMDA receptor and EGFR - the two proteins can physically associate in complexes. Meanwhile, CaMKII participates in a loop with Wnt signaling that affects TCF transcription factors in colorectal cancer cells. So blocking NMDA upstream can cascade down through multiple pathways to suppress several cancer-driving genes simultaneously.
The researchers caution that these conclusions are based on mRNA expression data only, not protein-level measurements. Gene expression changes don't always translate directly to protein activity changes, and the proposed pathway interactions are exploratory hypotheses rather than definitively proven mechanisms. Western blot or ELISA experiments would be needed to confirm these effects at the protein level.
This study provides the first detailed in vitro evidence that ketamine, at micromolar concentrations, kills HT-29 colorectal cancer cells through apoptosis and suppresses key cancer-driving genes. The convergence of cell biology data, gene expression results, and molecular docking from two computational methods strengthens confidence in the findings.
The clinical implication being explored is whether ketamine, already given to colorectal cancer patients as an anesthetic during surgery, might provide an additional anti-tumor benefit during and after the operation. This would be particularly appealing because it would require no new drug - just strategic use of an existing one. However, the authors strongly emphasize this possibility requires caution: in vitro results often don't translate directly to living organisms with complex immune environments and pharmacokinetics.
Key next steps identified include: testing multiple colorectal cancer cell lines (not just HT-29), running in vivo animal studies, investigating S-ketamine (the enantiomer with higher NMDA receptor affinity) separately, validating gene expression changes at the protein level, and conducting formal molecular dynamics simulations to better understand ketamine-receptor binding over time. The authors also note that racemic ketamine was used here, and S-ketamine might show even stronger effects given its higher NMDA affinity.