Investigating Cancer Tumor Growth Inhibition by Metformin and Therapeutic Ultrasound

Background：Metformin is the most widely prescribed drug worldwide for the treatment of type 2 diabetes. Recently, researchers have found that metformin has anti-cancer properties and it selectively targets cancer stem cells. In addition, therapeutic ultrasound has been extensively studied on tumor treatment. Ultrasound can induce both thermal effects and mechanical effects to destroy tumor microenvironment. Purpose：In this study, we investigated cancer tumor treatment by the combination of metformin and therapeutic ultrasound. Materials and Methods：In in-vitro study, CT-26 cancer cells and 96 well dish were used and each well was cultivated with 5 x 103 cells. There were four groups: Control, Met (1mM metformin for 24 hr), pUH (42℃, 0.94 MHz, 0.1 mW and 10% duty cycle ultrasound for 15 min), and pUH+Met. In in-vivo study, BALB/c male mice were used and the mice were injected subcutaneously with CT-26 Luc colon carcinoma cells in the right flank. Treatments were started when the tumors grew up to 50 mm3. The mice were randomly divided into four groups：Control, Met, pUH and Met+pUH. For the metformin treatment, the mice were given a dose of 25mg/kg metformin by oral gavage. For the ultrasound treatment, a frequency of 1 MHz, a duty cycle of 50%, a power of 2.7 W and a duration of 15 min were used. Tumors were measured every two days and H&E staining was used to study the effects of metformin and ultrasound on tumor tissues. Results：In in-vitro study, metformin plus therapeutic ultrasound decreased the cell viability of CT-26 to 60% for a single treatment. It was significantly different as compared to the other three groups. In in-vivo study, the treatment with metformin plus therapeutic ultrasound could effectively suppressed the tumor growth and the experimental results showed that there was significantly different as compared to the other three groups in tumor growth response after four treatments. Lymphocytic infiltration and inflammation could be observed on the H&E staining for the pUH and Met+pUH groups, and tissue rupture was obvious for these two groups. Summary：Metformin combined with therapeutic ultrasound could inhibit cancer cells and tumor growth significantly in the in-vitro and in-vivo studies. H&E staining showed the lymphocytic infiltration, inflammation and tissue disruption, indicating the thermal and mechanical effects of ultrasound sonication on tumor tissues. The results indicate that the effective treatment may come from both the damage by ultrasound sonication and the molecular mechanism caused by metformin. Keywords：Metformin、Therapeutic ultrasound、Tumor growth inhibition