Colorectal cancer is the most common gastrointestinal malignancy and a leading cause of cancer-related death worldwide. Chemotherapy remains the cornerstone of treatment, and 5-fluorouracil (5-FU) is the most widely used first-line drug, typically combined with oxaliplatin or irinotecan.
A major clinical obstacle is that cancer cells frequently develop resistance to 5-FU after repeated treatment cycles - often within 3 to 6 months. Once resistance develops, the drug loses its effectiveness and patients are left with limited options, especially in metastatic disease.
The underlying mechanism of this resistance involves multidrug resistance (MDR), which is driven largely by proteins in the ATP-binding cassette (ABC) transporter family. These proteins act as molecular pumps that actively expel chemotherapy drugs out of cancer cells before the drugs can do damage. The most important of these are MDR1 (ABCB1/P-gp) and MRP1 (ABCC1), both of which are frequently overexpressed in resistant tumors.
Finding compounds that can bypass or reverse this resistance is a critical priority. This study investigated whether anlotinib, a drug approved for other cancers, could re-sensitize 5-FU-resistant colorectal cancer cells to chemotherapy.
Anlotinib is a novel oral multi-target tyrosine kinase inhibitor (TKI) that simultaneously blocks multiple molecular targets involved in tumor growth and blood vessel formation. Its main targets include VEGFR2/3 (vascular endothelial growth factor receptors), FGFR1-4 (fibroblast growth factor receptors), PDGFR (platelet-derived growth factor receptor), and c-kit.
By blocking these receptors, anlotinib disrupts the signaling pathways that cancer cells use to proliferate, invade surrounding tissue, spread to other organs, and build new blood vessels that feed tumor growth. It has already been approved in China for advanced non-small-cell lung cancer and soft tissue sarcoma.
In colorectal cancer specifically, a clinical trial (the ALTER0703 trial) demonstrated that anlotinib monotherapy significantly prolonged progression-free survival as a third-line treatment in metastatic colorectal cancer. Other trials showed improvements when anlotinib was combined with chemotherapy in first-line metastatic colorectal cancer treatment.
Despite this clinical evidence, the precise mechanism by which anlotinib works in colorectal cancer cells - and in particular whether it can reverse acquired 5-FU resistance - had not been studied in detail. This study was designed to fill that gap.
The researchers used two human colorectal cancer cell lines that had been specifically engineered or selected to be resistant to 5-FU: HCT-8/5-FU and HCT-15/5-FU. These resistant lines were maintained in culture medium containing 5-FU at concentrations that keep them resistant, allowing the researchers to test new treatments under realistic resistance conditions.
To measure how effectively anlotinib and 5-FU killed cells, the team used the MTS assay, a colorimetric test that measures cell metabolic activity as a proxy for cell viability. Cells were treated with varying drug concentrations for 24 and 48 hours, enabling calculation of IC50 values (the concentration required to inhibit cell growth by 50%).
Colony formation assays were used to test long-term effects. Unlike MTS, which measures short-term metabolic activity, colony assays assess whether cells can continue to divide and form new colonies over 12 days - a measure of sustained proliferative capacity more relevant to tumor regrowth and treatment durability.
To study how anlotinib affects the cell cycle (the stages cells pass through during division), the researchers used flow cytometry with propidium iodide staining. Finally, immunoblotting (western blot) was used to measure protein levels of MDR1, MRP1, total AKT, and phosphorylated AKT (p-AKT), which reflects how active the AKT survival pathway is.
A key first finding was that while both cell lines were highly resistant to 5-FU (with IC50 values of over 2,000 micromolar for HCT-8/5-FU and about 18 micromolar for HCT-15/5-FU at 48 hours), both remained sensitive to anlotinib. The IC50 of anlotinib ranged from approximately 8 to 55 micromolar depending on cell line and time point.
When anlotinib was added to 5-FU at sub-cytotoxic doses (IC5 and IC10 - concentrations that cause only minimal cell death on their own), the combination produced a significantly greater inhibition of cell growth than either drug alone in MTS assays. This indicates that even very low concentrations of anlotinib can meaningfully re-sensitize resistant cells to 5-FU.
The colony formation assays revealed an even more striking result. Anlotinib at IC10 concentration showed minimal acute cytotoxicity in short-term MTS assays, but it dramatically reduced the number and size of colonies when combined with 5-FU. This discrepancy shows that anlotinib's impact on long-term proliferative capacity is far greater than its immediate toxicity suggests.
This difference between short-term and long-term assay results is important clinically: it implies that anlotinib may work by causing durable growth arrest or impairing the cancer cells' ability to self-renew, rather than by directly killing them immediately. These persistent effects on proliferation are more relevant to preventing tumor regrowth.
Flow cytometry revealed that anlotinib induced a dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle, meaning cells became stuck at the resting or preparation stage rather than progressing into active DNA synthesis (S phase). This effect was most pronounced at 20 micromolar in HCT-15/5-FU cells and 10 micromolar in HCT-8/5-FU cells.
Importantly, anlotinib did not significantly increase apoptosis (programmed cell death) at the doses tested. This suggests that anlotinib's primary mechanism of action is cytostatic (stopping cell division) rather than cytotoxic (killing cells outright). By trapping cells in G0/G1, anlotinib prevents them from replicating, which may render them more vulnerable to 5-FU's interference with DNA synthesis when the cells eventually try to progress.
Western blot analysis showed that MDR1 and MRP1 protein levels were significantly reduced in both resistant cell lines as anlotinib concentration increased. Crucially, this downregulation was visible even at IC5 and IC10 concentrations - the same very low doses used in the sensitization experiments - providing a direct molecular explanation for the observed reversal of 5-FU resistance.
At the same time, phosphorylated AKT (p-AKT) levels decreased in a concentration-dependent manner while total AKT remained unchanged. Since the AKT signaling pathway is known to regulate ABC transporter expression, this finding suggests that anlotinib may downregulate MDR1 and MRP1 at least partly through inhibition of AKT phosphorylation.
The convergence of anlotinib's effects on both MDR transporters and AKT signaling is mechanistically significant. Emerging evidence indicates that the PI3K/AKT pathway can regulate ABC transporter expression through transcriptional and post-translational mechanisms. Therefore, anlotinib's suppression of AKT phosphorylation may be the upstream event that leads to downregulation of MDR1 and MRP1.
By reducing MDR1 and MRP1 - the molecular pumps that expel 5-FU from cancer cells - anlotinib may increase intracellular accumulation of 5-FU, allowing the drug to reach effective concentrations inside the cell where it can interfere with DNA synthesis and cell division. This would explain why 5-FU becomes dramatically more effective when combined with sub-cytotoxic anlotinib doses.
The cell cycle arrest in G0/G1 adds another dimension. By slowing cells down before they enter S phase (active DNA replication), anlotinib may prime resistant cells to be more vulnerable to 5-FU's anti-metabolite action. 5-FU works by disrupting DNA and RNA synthesis, processes that are active during S phase - so cells on the verge of entering S phase may be especially susceptible to 5-FU's effects once anlotinib has reduced their resistance machinery.
This multi-pronged mechanism - reducing drug efflux pumps, inhibiting AKT signaling, and inducing cell cycle arrest - collectively provides a strong rationale for combining anlotinib with 5-FU in patients who have developed 5-FU resistance, rather than relying on either drug alone.
The concentrations of anlotinib at which sensitization effects were observed in this study (IC5 and IC10) are within the range of clinically achievable plasma concentrations reported in pharmacokinetic studies of patients receiving anlotinib. This means the doses needed to re-sensitize resistant cells in the lab are not unrealistically high - supporting the real-world translational potential of these findings.
The fact that these effects occur at sub-cytotoxic doses is clinically important because it suggests that anlotinib could be added to 5-FU-based regimens without substantially increasing toxicity. This is especially valuable for patients with metastatic colorectal cancer who have already been through multiple lines of therapy and have limited tolerance for additional treatment-related side effects.
Anlotinib already has an established safety profile from its approved uses in lung cancer and sarcoma, and the ALTER0703 trial has demonstrated that it prolongs progression-free survival in metastatic colorectal cancer as a third-line treatment. The current mechanistic findings provide a rationale for specifically targeting the subgroup of patients who have acquired 5-FU resistance, where the combination may offer the greatest benefit.
These results support the design of future clinical trials investigating low-dose anlotinib in combination with 5-FU-based chemotherapy in patients with refractory colorectal cancer, offering a potential strategy to improve outcomes in a population with very limited therapeutic options.
The authors acknowledge important limitations of this study. It is an in vitro study only, meaning it was conducted entirely in cell cultures, not in living organisms. In vitro findings do not always translate to in vivo efficacy, and questions about pharmacokinetics, tumor microenvironment effects, and toxicity in a whole organism cannot be answered from cell culture data alone.
The study did not conduct formal synergy analysis using established methods such as the Chou-Talalay combination index, so the nature of the drug interaction (whether truly synergistic or merely additive) was inferred qualitatively from the experimental data rather than quantified precisely.
Only two resistant cell lines were studied, and the mechanistic analysis focused primarily on MDR transporters and AKT signaling. Other pathways that may contribute to anlotinib's effects - such as MAPK/ERK, Wnt/beta-catenin, or autophagy - were not investigated, leaving open the possibility that additional mechanisms are at play.
Future work should employ animal xenograft models to test the in vivo efficacy and safety of anlotinib-5-FU combinations, incorporate formal synergy analysis, examine a broader range of dosing combinations, and ultimately validate these findings in clinical specimens from patients with acquired 5-FU resistance.