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Introduction: Bitter melon extract (BME) is known to exhibit cytotoxic effects on breast cancer cells (MCF-7). However, the molecular mechanisms by which BME exerts cytotoxic effects are not established. Aims: We aimed to investigate if BME exerts cytotoxic effects on MCF-7 cells via inhibiting Rho kinase (ROK) activity and Rhotekin expression. Both TOK and Rhotekin mediate several cellular processes such as cell division, attachment, migration etc. We hypothesized that BME exerts cytotoxic effects on MCF-7 cells by decreasing ROK activity and Rhotekin expression. Methods: Fresh bitter melons were purchased from an Asian grocery store and the extract (BME) was extracted, centrifuged, and filter sterilized. The MCF-7 cells were cultured in DMEM medium with 1% BME (v/v). After culturing cells for 48 hours, pictures of cultures were taken to confirm cytotoxic effects on cells. In our earlier experiments, BME induced decrease in cell viability was also confirmed using Trypan blue exclusion and MTT assays. The cells were harvested and changes in the protein expression of phospho-MYPT1 and Rhotekin were determined using anti-phospho-MYPT1 and anti- Rhotekin antibodies in the western blotting. Since ROK directly phosphorylates MYPT1, reduced expression of phospho-MYPT1 would indirectly suggest reduced ROK activity. Similarly, reduced expression of rhotekin is a downstream mediator of ROK activation pathway and reduced level of rhotekin would also contribute to the reduced viability of cells. Results & Discussion: BME exerted cytotoxic effects on MCF-cells (N=4). However, BME did not decrease expression levels of phospho-Thr 853-MYPT1 (N=3, duplicate gels). Also, BME did not affect Rhotekin expression (N=3, duplicate gels). Three different loading control proteins (actin, myosin lighta chain, and GAPDH) were used and none of them were affected by the BME. The data together rejects our hypothesis. In the future, we will be running more sets of experiments to confirm our findings. FRD-MUCOM. All authors contributed equally to this work. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.