Colorectal cancer (CRC) is the third most common cancer worldwide, with nearly 1.9 million new cases diagnosed in 2020 and approximately 0.94 million deaths. It is the second leading cause of cancer-related death globally. Despite improvements in screening programs and treatment options, the 5-year survival rate for advanced CRC remains below 15%, reflecting the serious challenge this disease poses to patients and families.
A major reason for the poor survival rate is drug resistance. Over time, cancer cells develop mechanisms to survive even in the presence of chemotherapy drugs, causing treatments that initially worked to gradually lose their effectiveness. Nearly a quarter of patients who undergo potentially curative surgery for CRC later develop new tumors (metachronous metastases), and more than 25% of newly diagnosed patients already have metastatic disease at diagnosis. These challenges highlight the urgent need for new therapeutic combinations that can overcome resistance and treat cancer more effectively.
One strategy researchers are exploring is drug repurposing, which involves testing drugs already approved for other conditions to see if they also work against cancer. This approach can save time and money compared to developing entirely new drugs from scratch. Imatinib (IM), a drug originally developed to treat certain types of leukemia and gastrointestinal stromal tumors by blocking enzymes called tyrosine kinases, has shown promising effects in colorectal cancer in laboratory studies.
Another promising compound is Zebularine (ZEB), a drug that works by disrupting DNA methylation, a process where chemical tags are added to DNA to silence certain genes. In cancer, DNA methylation is often abnormal, causing important tumor-suppressor genes (genes that would normally halt cancer growth) to be silenced. Zebularine can reverse this silencing, potentially reactivating protective genes and making cancer cells more vulnerable to other treatments. The potential to combine Imatinib and Zebularine to achieve a stronger anti-cancer effect than either drug alone is the central question of this study.
All experiments in this study were conducted using HCT-116 cells, a well-established human colorectal carcinoma cell line known for its aggressive behavior, limited differentiation, and ability to model metastatic colon cancer in the laboratory. The cells were grown in standard culture conditions and treated with Imatinib (IM), Zebularine (ZEB), or both drugs together. Initial experiments tested a range of concentrations to establish the IC50, the concentration needed to kill 50% of the cells, for each drug individually.
To determine whether the two drugs interact synergistically, additively, or antagonistically, the researchers used CompuSyn software to calculate a combination index (CI). A CI below 1.0 indicates synergy, meaning the drugs together are more effective than the sum of their individual effects. A CI above 1.0 indicates antagonism. The combination of 4 micromolar (uM) Imatinib and 20 uM Zebularine was selected for detailed mechanistic studies based on its favorable synergy profile.
Multiple laboratory techniques were then used to understand how the combination works. Flow cytometry was used to measure both cell cycle changes and programmed cell death (apoptosis). Quantitative PCR (qPCR) measured changes in the expression of key genes involved in cancer cell survival, metastasis, and apoptosis. Western blotting assessed protein levels for survivin, P-glycoprotein, Wnt, and SIRT1, all proteins critical to cancer drug resistance and growth. An ELISA-based assay quantified levels of EGFR, a receptor often overactive in colorectal cancer.
To assess cancer cell migration (a key step in metastasis), a scratch wound healing assay was performed by creating a gap in a layer of cancer cells and measuring how quickly the cells migrated to fill the gap under different treatment conditions. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure exactly how much of each drug was being taken up by the cancer cells, testing whether one drug might affect the cellular absorption of the other.
Both Imatinib and Zebularine individually inhibited the growth of HCT-116 colorectal cancer cells in a concentration-dependent manner. The IC50 for Imatinib was 17 uM and for Zebularine was 40 uM when tested alone. However, when combined, the IC50 of Zebularine dropped dramatically from 40 uM to 20 uM, even when using only one-quarter of the Imatinib IC50 dose (4 uM). This halving of the Zebularine dose needed to achieve the same effect already suggests a beneficial interaction.
Formal synergy analysis using the combination index method confirmed a CI of 0.61 for the 4 uM Imatinib plus 20 uM Zebularine combination, clearly indicating synergy (any CI below 1.0 is considered synergistic). This means the combination killed far more cancer cells than would be predicted from simply adding the effects of each drug separately. The dose reduction index (DRI) was greater than 1 for both drugs, meaning the synergistic combination allows effective cancer cell killing at doses lower than would be required for either drug alone, potentially reducing toxicity in future clinical use.
When cells were treated with the combination, the percentage of cells arrested in the S-phase of the cell cycle (the DNA replication phase) jumped to 69.8%, compared to 32.31% for Imatinib alone, 25.93% for Zebularine alone, and only 19.20% in untreated cells. Arresting cells in S-phase prevents them from completing division and producing new daughter cancer cells, effectively halting tumor growth. This dramatic increase confirms the combination profoundly disrupts cancer cell proliferation.
The combination also increased cancer cell death. Both apoptosis (programmed, controlled cell death) and necrosis (a more explosive type of cell death) were elevated in the combination group. Notably, necrotic percentages in all treatment groups (approximately 70-74%) were dramatically higher than in untreated cells (3.3%), confirming that the treatments were highly effective at inducing cell death through multiple pathways.
To understand the mechanisms driving the synergistic cell killing, the researchers measured changes in the expression of multiple key genes and proteins. The combination treatment caused significant downregulation (reduction) of MMP9 and MMP2, two enzymes that colorectal cancer cells use to break down tissue barriers and spread to other parts of the body (metastasis). By reducing these metastasis-enabling enzymes, the combination therapy may help contain the cancer and prevent it from spreading.
At the same time, the combination strongly upregulated Caspase-9, a key protein that activates the internal (intrinsic) pathway of programmed cell death (apoptosis). The anti-apoptotic protein Bcl-2, which normally acts as a survival signal protecting cancer cells from death, was significantly downregulated by the combination. These changes in apoptotic regulators confirm that the combination treatment actively pushes cancer cells toward self-destruction through well-defined biological pathways.
The combination reduced EGFR (epidermal growth factor receptor) levels by 55%, compared to 25% reduction with Imatinib alone and 18% with Zebularine alone. EGFR is a protein on the surface of many cancer cells that drives their growth and survival when activated. Overactive EGFR is a major driver of colorectal cancer progression and is already a validated target for existing CRC drugs like cetuximab. The fact that this combination achieves a much greater EGFR reduction than either drug alone represents a significant potential advantage.
The combination also caused marked downregulation of four important proteins linked to cancer drug resistance and progression: survivin (a protein that prevents cancer cell death and promotes tumor growth), P-glycoprotein (P-gp) (a protein that pumps chemotherapy drugs out of cancer cells, a major cause of multidrug resistance), Wnt (a signaling protein driving colorectal cancer growth), and SIRT1 (a protein that promotes Wnt signaling). Simultaneously reducing all four of these resistance and growth factors suggests the combination targets multiple pathways at once, potentially making it harder for cancer cells to develop resistance.
One particularly interesting mechanistic finding was that Imatinib appears to help more Zebularine enter the cancer cells. Using precise LC-MS/MS measurements, the researchers found that when the two drugs were combined, the intracellular levels of Zebularine were significantly higher than when Zebularine was given alone (p = 0.01). In contrast, combining the drugs did not significantly change how much Imatinib was taken up by the cells. This one-way interaction helps explain part of why the combination is more effective.
The mechanism likely involves organic cation transporters (hOCTs), proteins that sit on the cell surface and control what chemicals enter and exit cells. Imatinib may reduce the activity of transporters that normally pump Zebularine back out of cells (efflux transporters), allowing more Zebularine to accumulate inside the cancer cell. Higher intracellular Zebularine concentrations mean more effective DNA methylation inhibition and greater reactivation of silenced tumor-suppressor genes, amplifying the anti-cancer effect.
The oxidative stress effects of the combination also contributed to cancer cell death. The combination treatment increased malondialdehyde (MDA) by 20% and nitric oxide (NOx) by 105.8% compared to Imatinib-treated cells, while simultaneously decreasing protective glutathione (GSH) by 34.9%. MDA and NOx are markers of oxidative damage inside cells, while GSH is an antioxidant that cancer cells use to protect themselves. This shift creates a highly toxic internal environment for cancer cells that promotes multiple types of cell death.
The combination's ability to downregulate P-glycoprotein is also clinically significant. P-glycoprotein is one of the most important causes of multidrug resistance in cancer treatment. When cancer cells overexpress P-gp, they physically pump many different chemotherapy drugs out of the cell before those drugs can do their work, rendering treatments ineffective. Reducing P-gp expression could make cancer cells more sensitive not just to Imatinib and Zebularine, but potentially to other chemotherapy agents used alongside them.
Cancer metastasis, the spread of cancer to other parts of the body, is responsible for the majority of colorectal cancer deaths. Cell migration, the ability of cancer cells to physically move and invade new tissues, is a prerequisite for metastasis. The scratch wound healing assay measured how well cancer cells migrate to close a gap in a cell monolayer. Untreated cells showed approximately 90% wound closure within 48 hours, reflecting very strong migratory activity.
Both drugs individually reduced wound closure to approximately 65-70%, representing a meaningful reduction in cancer cell migration. The combination of Imatinib and Zebularine reduced wound closure further to approximately 60%, the lowest of all treatment conditions. While the difference between combination and individual treatments did not reach statistical significance in this experiment, the trend is consistent with the broader anti-metastatic effects seen in the molecular analyses, particularly the reductions in MMP9 and MMP2.
For patients and families, the significance of these findings is in what they suggest about future treatment possibilities. The ability of this combination to simultaneously reduce cancer cell growth, trigger cell death through multiple mechanisms, suppress metastasis-enabling proteins, reduce EGFR (a drug target already validated clinically), and lower P-glycoprotein levels (a resistance mechanism) makes it a scientifically compelling candidate for further development. These diverse effects might make it harder for cancer cells to develop resistance compared to single-agent therapy.
It is important to note that this study was conducted entirely in laboratory cell culture (in vitro). Results from cell culture experiments do not always translate directly to what happens in living organisms or in human patients. Before this combination could be tested in patients, it would need to undergo preclinical animal studies to evaluate its safety and efficacy in a living system, followed by clinical trials to assess its benefit and safety in humans. The authors acknowledge this limitation and call for further research in preclinical and clinical settings.
This study demonstrates that combining Imatinib and Zebularine produces synergistic anti-cancer effects in human colorectal cancer cells, achieving greater cancer cell killing at lower drug doses than either drug alone. The combination index of 0.61 mathematically confirms synergy, and the broad range of molecular changes observed, spanning cell death, cell cycle arrest, metastasis suppression, EGFR reduction, and resistance protein downregulation, suggests the combination attacks colorectal cancer cells through multiple complementary pathways simultaneously.
The identification of the Wnt-Survivin-P-Glycoprotein pathway as a key target of this combination is particularly noteworthy. This pathway sits at the intersection of cancer cell survival, tumor growth signaling, and drug resistance. Targeting it through the Imatinib-Zebularine combination could help overcome one of the most intractable challenges in colorectal cancer treatment: the tendency of cancer cells to develop resistance to any single drug over time.
The finding that Imatinib increases the intracellular uptake of Zebularine provides a rational pharmacological basis for the synergy, suggesting the two drugs enhance each other's cellular delivery as well as their direct anti-cancer effects. Combined with the oxidative stress enhancement and the reduction in protective glutathione, this creates a multilayered attack on cancer cell defenses.
This research adds to a growing body of evidence supporting the strategy of combining epigenetic drugs like Zebularine with targeted therapies like Imatinib to overcome resistance and improve outcomes in hard-to-treat cancers. While the findings are promising, they represent an early step in a research pipeline that would need to proceed through animal models and ultimately clinical trials before any benefit to patients can be claimed. The authors call for further studies to confirm these findings and evaluate the combination in more complex biological systems.