Inhibitory Effect Of Gynostemma Pentaphyllum Combined With 5-Fu On The Growth Of Colorectal Cancer

Colorectal cancer (CRC) is the third most common malignant cancer second only to lung cancer and bearst cancer in the world, which often occurs at the colon or rectum of the human digestive tract and be considered as the most lethal gastrointestinal tumors. 5-Fluorouracil (5-Fu) is one of the most widely used chemotherapeutic drug for treatment colorectal cancer. The effect of inhibition of 5-Fu to cancer cells mostly related to the drug dosage that cause severe toxic- and side-effects on normal cells, and gradually increasing drug resistance also limits its dosage and therapeutic effect. Gypenosides (Gyp) is the major medicinal components extracts from Gynostemma pentaphyllum (Thunb.) Makino. Experimental study has reported that Gyp could significantly inhibit the growth of a variety of colorectal cancer cells and trigger apoptosis. The present paper exolores the effects of Gyp combined with 5-Fu on colorectal cancer cell growth, apoptosis and cycle progression, analysis the possible molecular mechanisms pathway, and through the establishment of tumor-bearing model to verify the anti-tumor effects of Gyp+5-Fu, disscuss the safety of both combination therapy. The results are as follows:1. The inhibition effect of Gyp combined with 5-Fu on S W-480 and Caco2 cells.When the drug dose in between 1?50 ?g/ml,5-Fu could causes the decrese of cell viability of SW-480 and Caco2 in a time-dependent manner pathway. Gyp (drug dose in between 30?50 ?g/ml) inhibited SW-480 and Caco2 cell proliferation in a dose-and time-dependent manner. The IC50 value was calculated as 99.59 ?g/ml,112.22 ?g/ml for SW-480 and Caco2 cells after incubation for 24 h. When co-treated cells with Gyp (70,85,100 ?g/ml) and 5-Fu (5,10 ?g/ml) simultaneously, the combined treatment even in the low doses displayed much higher anti-proliferative activities on SW-480 and Caco2 cells than that of single treatment.2. Apoptotic response and cell cycle distriduction induced by Gyp and 5-Fu on SW-480 cells.Flow cytometry analyising and annexin V-PE/7-AAD staining indicates 5-Fu+ Gyp could induce SW-480 cell apoptosis. Hoechst 33342 staining showed that cells exerted remarkably cell apoptosis changes:condensed chromatin, fragmented punctuate blue nuclear fluorescence. Western blotting was performed to analysis the potential mechanism worked in 5-Fu and Gyp triggered apoptosis. Results showed that 5-Fu+ Gyp greatly enhanced caspase 3, caspase 9 and PARP cleavage and down-regulated the expression of anti-apoptotic protein Bcl-2 in SW-480 cells. Gyp was also found to augment 5-Fu-induced G0/G1 phase arrest. And 5-Fu+Gyp treatment remarkably increased the expression of phospho-p53, but decreased the expression of cyclin E and phospho-cdk2.3. The effects of ROS-induced DNA damage and p53 activation after Gyp+5-Fu treatment in SW-480 cells.Single Gyp or 5-Fu treatment can induce DNA damage in SW-480 cells. DNA may be the common molecular targets for Gyp and 5-Fu treatment. The production of ROS and p53 activation plays an important role in Gyp and 5-Fu co-treatment. Comprehensive analysis of experimental results, the effects of Gyp combined with 5-Fu on SW-480 cells'apoptosis and cycle arrest and a series of reactions of fundamental signaling pathways can be summarized as follows:5-Fu causes DNA damage by its misincorporation, whereas Gyp synergistically induces intracellular ROS generation through regulation of the redox system and thus enhanced the 5-Fu-induced DNA damage, resulting in activation of the p53 activation, which in turn induces cell cycle arrest in G0/G1 and triggers mitochondrial dysfunction to amplify the apoptotic signals.4. The anti-tumor effect of Gyp and 5-Fu on CT-26 tumor-bearing mice.Gyp alone could significantly inhibit the growth of tumor volume. And the tumor inhibition rates was 44.21% for 25 mg/kg Gyp treatment.5-Fu and Gyp in combination exhibits much superior tumor volume and weight inhibition on CT-26 xenograft mouse model in comparison to 5-Fu or Gyp alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation. Analysis of preliminary toxicological results discovery,5-Fu+Gyp treatment can achieve good anti-tumor effect while avoiding the toxicology to body's liver and kidney by a high concentration of 5-Fu treatment, and obtain a very good effect of drug combination therapy.