Effects of Hydroalcoholic Extracts of Rosemary (Rosmarinus officinalis) Leaves and Aerial Parts on the YM-۱ Esophageal Cancer Cell Line

Esophageal carcinoma is the seventh largest cause of cancer death worldwide. The two main pathological categories are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) (Yang and colleagues, ۲۰۲۴). Recent epidemiological studies have demonstrated a persistent increase in the incidence of ESCC worldwide. ESCC is often treated with radiotherapy. Radiation-induced DNA damage may induce the release of cytokines and chemokines, generating inflammatory responses and alterations in the tumor microenvironment, limiting immune function, and leading to ESCC invasion and metastasis (An and colleagues, ۲۰۲۳). On the other hand, the concomitant application of radiotherapy and chemotherapy is linked to several limitations. A key concern is that the therapeutic doses of these treatment approaches are often limited by their non-selective toxicities, which can cause severe damage to healthy tissues surrounding the tumor (Villegas and colleagues, ۲۰۲۴). Consequently, there remains an ongoing need to advance therapeutic approaches for the treatment of esophageal cancer Various phytogenic compounds have been shown to be effective in preventing and treating multiple tumor types. Rosmarinus officinalis L. is a herb cultivated worldwide. There is growing interest in the medicinal properties of rosemary, which is used for its anti-inflammatory, anti-infective, and anticancer effects. The anti-tumor effect of rosemary is associated with various mechanisms, including antioxidant activity, anti-inflammatory activity, anti-angiogenic properties, epigenetic effects, modulation of the immune response, alteration of specific metabolic pathways, and enhanced expression of tumor suppressor genes (Allegra and colleagues, ۲۰۲۰). The anticancer properties of Rosmarinus officinalis against esophageal cancer have been previously documented (Sirajudeen and colleagues, ۲۰۲۴); however, this study characterizes the anticancer profile of Rosmarinus officinalis in the YM-۱ esophageal cancer cell line, a squamous cell carcinoma model that has not been investigated to date MethodsThe plant material (۳۰ g) was macerated in ۲۰۰ mL of ۸۰% methanol and left to remain at room temperature for ۴۸ h. The extracts were filtered, and the residue was re-extracted under the same conditions after ۲۴ hours. The second extract was added to the initial extract. Subsequently, the solvent was removed under a rotary evaporator at < ۴۰ °C, and the resulting extract was stored at ۴ °C for further investigation (Pourmorad and colleagues, ۲۰۰۶).The human esophageal cancer cell line YM-۱ was used in this study, obtained from the cell bank of the North Research Centre, Pasteur Institute of Iran. YM-۱ cells were cultured in low-glucose DMEM/F۱۲ supplemented with GlutaMAX, ۱۰% fetal bovine serum, and penicillin-streptomycin at ۳۷ °C in a humidified environment. Cells were passaged at approximately ۷۰% confluency (Khosravi and Asadi, ۲۰۲۱). Cell viability was measured by MTT colorimetric assay (۳-(۴,۵-dimethylthiazol-۲-yl)-۲,۵-diphenyltetrazolium bromide). Briefly, each well of a ۹۶-well culture plate was seeded with ۱×۱۰۴ cells of YM-۱. Cells were treated with rosemary extract at final concentrations of ۷۸.۱۲۵, ۱۵۶.۲۵, ۳۱۲.۵, ۶۲۵, ۱۲۵۰, and ۲۵۰۰ µg/mL for ۲۴, ۴۸, and ۷۲ h after ۲۴ h of incubation. MTT test was used to determine cell viability at the end of each treatment period, and the absorbance was measured at ۵۴۰ nm using a microplate reader (Kumar and colleagues, ۲۰۱۸). Cell viability was calculated by the following equation: Cell vitality (%) = (Mean absorbance of treated cells / Mean absorbance of control cells) × ۱۰۰Statistical analysis was performed using SPSS software, one-way analysis of variance (ANOVA) followed by Duncan's post hoc test. The data were expressed as mean ± standard deviation (SD) of three replicates. Differences were statistically significant at ۵% level of significance (p < ۰.۰۵).ResultsThe rosemary extract significantly decreased the viability of YM-۱ esophageal cancer cells in a time- and concentration-dependent manner. A significant reduction in cell viability was observed at ۷۸.۱۲۵ µg/mL and higher doses across all treatment periods. The lowest cell viability was observed at the highest extract concentration (۲۵۰۰ µg/mL), with values of ۱۰.۳۴ ± ۰.۶۹ %, ۱۰.۲۰ ± ۰.۸۸ %, and ۸.۳۶ ± ۰.۳۱ % at ۲۴, ۴۸, and ۷۲ h, respectively. Moreover, the cytotoxic effect was time-dependent, as the IC۵۰ decreased progressively over ۲۴, ۴۸, and ۷۲ h from ۵۱۶.۵۱ µg/mL to ۴۳۷.۷۳ µg/mL and ۳۵۸.۴۲ µg/mL, respectively Discussion and ConclusionIn the present work, it was shown that hydroalcoholic extracts from Rosmarinus officinalis leaves and aerial parts exhibit significant cytotoxicity against the YM-۱ ESCC cell line. The extract reduced cell viability in a dose- and time-dependent manner, and higher doses and longer incubation times exerted greater inhibitory effects. The time-dependent decline in IC۵۰ values further suggests that the extract's anticancer potential increases over time. These data show bioactive constituents of Rosmarinus officinalis with potent anti-proliferative activity against esophageal cancer cells, especially in an ESCC model. Collectively, the findings show the potential of rosemary as a feasible natural source for the development of innovative therapeutic modalities against esophageal cancer; however, further in vitro mechanistic and in vivo research are needed to establish its clinical use

Keywords:

Rosmarinus officinalis , Esophageal squamous cell carcinoma , YM-۱ cell line , MTT assay , Cytotoxicity