Promising anticancer activity of cromolyn in colon cancer: in vitro and in vivo analysis

The MCF-10 cell line, characterized as a normal epithelial cell line, and the HT-29 cell line, representing human colorectal adenocarcinoma cells, were acquired from the Pasteur Institute in Iran. Additionally, male BALB/c mice and the CT26 cell line were procured from the same source.

For cell culture, we employed DMEM medium and fetal bovine serum, both sourced from Gibco Corporation in the USA. Penicillin–streptomycin (0.1%) was acquired from Biosera Company in France. Dimethyl sulfoxide (DMSO) was prepared from materials provided by Merck in Germany, and the MTT reagent was sourced from Sigma in the USA.

We purchased Cromolyn from Sinadarou Pharmaceuticals in Iran and obtained Doxorubicin from Actoverco Pharmaceuticals, also based in Iran.

To assess apoptosis, we utilized the Annexin V/PI Apoptosis Assay Kit, which was obtained from Mab-Tag GmbH Company in Germany.

In the cell culture procedure, HT-29 cells were grown in high-glucose DMEM, while MCF-10 cells were cultured in DMEM. Both culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were maintained in a controlled environment at 37 °C with 5% CO2 for optimal growth conditions. To assess cell viability, Trypan blue staining was employed (Freshney 2015).

Cell proliferation was assessed using the MTT assay, a well-established technique. HT-29 and MCF-10 cells were seeded at a density of 10^4 cells per well in 96-well microplates, reaching 80% confluence. They were then incubated at 37 °C in a 5% CO2 environment. We added different concentrations of Cromolyn, ranging from 0.5 to 16 µM, and Doxorubicin, used as a positive control drug with concentrations of 1, 2, 4, 8, 16, and 32 nM, to the wells containing fresh media. This replacement was carried out after 24 h, while the negative control wells were filled with fresh media containing only DMSO. Following this, the microplates were incubated for 72 h in a controlled environment at 37 °C with 5% CO2. Subsequently, 10 µL of MTT reagent at a concentration of 0.5 mg/mL was added to each well, and the incubation continued in darkness for 4 h. After removing the supernatant, 150 µL of DMSO was introduced into each well to serve as a formazan solvent. The microplates were then agitated for 20 min on a shaker. Optical density (OD) at a wavelength of 570 nm was measured for each well using an ELISA reader. These experimental procedures were carried out in triplicate. The percentage of viable cells was calculated using the formula: 100—(OD treatment / OD control) × 100 (Freshney 2015).

IC50 values, representing the concentration at which cell proliferation is inhibited by 50%, were determined using CurvExpert 1.4 software. The selectivity index (SI), which assesses compound safety, was calculated using the formula: Selectivity Index = IC50 in normal cells / IC50 in cancer cells. A compound with an SI greater than 2 is considered selective (Demirgan et al. 2016; Zbakh et al. 2020).

To evaluate the impact of Cromolyn and DOX on apoptosis in HT-29 and MCF-10 cell lines, we utilized the Annexin V/PI Apoptosis Assay Kit. Initially, the cells were seeded in 24-well plates and allowed to incubate overnight at 37 °C in a 5% CO2 environment. The following day, the culture medium was replaced with a fresh medium containing IC50 concentrations of Cromolyn and DOX, and this incubation was sustained for 48 h. Subsequently, the cells were transferred to flow cytometry tubes and washed with PBS. In each tube, a mixture of 100 µL of binding buffer, 5 µL of Annexin-V, and 5 µL of PI was added, followed by a 20-min incubation in darkness. Following incubation, 400 µL of binding buffer was introduced, and the tubes underwent centrifugation at 400 g for five minutes. Finally, the cells were resuspended in an additional 500 µL of binding buffer before being analyzed using flow cytometry software. (Adan et al. 2017).

For this study, we utilized a total of thirty male BALB/c mice, each within the same age range and weighing between 20 to 25 g. The CT26 cells were cultured, and subsequently, we introduced 2 × 10⁶ cells suspended in 10µL of PBS via subcutaneous injection into the left flank of three mice. Three weeks later, when the tumors had grown to a size of 1,500 mm³, they were surgically excised, and 5 mm³ sections were prepared by punching. These sections were then transplanted subcutaneously into the left side of 18 other mice.

Two weeks following the transplantation, when the tumors had reached a size of 100 mm³, we divided the mice into three groups, each consisting of six individuals. The Cromolyn group received Cromolyn intraperitoneal (IP) at a dose of 50 mg/kg every other day for a period of 35 days, equivalent to 0.05 LD50 of the drug. Similarly, the DOX group received DOX at a dose of 558 µg/kg IP every other day for 35 days, also equivalent to 0.05 LD50. The LD50 dose of the drug was determined based on data extracted from past articles mentioned on the https://​pubchem.​ncbi.​nlm.​nih.​gov website.

The Cromolyn group received Cromolyn intraperitoneal (IP) at a dose of 50 mg/kg, equivalent to 0.05 × LD50 of the drug. Katzung and Trevor’s textbook Pharmacology states that therapeutic doses in humans are equivalent to 0.01–0.10 (mean 0.05) LD-50 in animals. Thus, we chose this dose.

The negative control group was administered normal physiological saline. Upon the death of each mouse, we removed the tumors, and a pathologist verified the tumor tissues after preparing slides (Xu et al. 2017; Aliabadi et al. 2023).

We measured the weight of the tumors using a digital scale and assessed their dimensions with calipers to determine their small and large diameters: