The Research Of The Combination Treatment Of Intrahapetec Cholangiocarcinoma Cells With TRAIL And Gemcitabine

Intrahepatic cholangiocarcinoma is a kind of primary liver cancer. When diagnosed, most of patients often have already been late stage. Lost the chance of surgical resection. Still chemotherapy, radiotherapy, and immunotherapy are not very sensitive, too. So,we have no effective therapeutic method at present. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) is a new chemotherapeutics which can induce tumor cell apoptosis, because of it's features of preferentially kill tumor cells but do no harm to normal cells, so it has became a hot reaserch topic. Lately reaserch shows that some tumor cells perform drug tolerance to TRAIL, however, these tumors could be re-sensitive to TRAIL by combined treatment with chemotherapy drugs. So combinated treatment with TRAIL and chemotherapy could become a new method of tumortherapy.We use intrahepatic cholangiocarcinoma cell line (HCCC-9810) as experimental subject.Firstly, we set up five different consistence TRAIL groups (10,30,100,300,1000ng/ml),we use MTT test the growth inhibition rate and SPSS 13.0 analyse the result. We found out that when 100ng/ml and above concentration shows significant difference with control group(P< 0.01), Also the growth inhibition rate amang 100,300,1000ng/ml show no statistical difference (P>0.05).So we can conclude that small does HCCC-9810 was not very sensitive to TRAIL when used alone,and IHCC-9810 perform drug tolerance to TRAIL.So we took 100ng/ml TRAIL undertake next experiment step. The gemcitabine group (10,20,40,80μg/ml) shows that the growth inhibition rate rely on both time and drug concentration.The growth inhibition rate of combined TRAIL (100ng/ml)with different concentration gemcitabine (10,20,40,80μg/ml) shows that the combination group can minificantly kill more tumor cells than both TRAIL and gemcitabine group. We can see in combined group, gemcitabine and TRAIL has coordination effect on HCCC-9810. Thus, used alone, TRAIL perform drug tolerance to HCCC-9810.After gemcitabine process,HCCC-9810 cells can be re-sensitive to TRAIL.Then we use Hoechst33258 fluorescence staining techniques, found out combined group cell appeared typical cell apoptosis form:nuclear gathered, marginalized, nuclear chipped.For TRAIL and gemcitabine group,there were no obvious signs of apoptosis.It can be concluded that the TRAIL joint gemcitabine group kill tumor cells by inducing apoptosis.Further more, we detected the apoptosis from protein level by using western-blot method,we detected the expression levels of Caspase-8, Caspase-3 and their cleavages in each group, we only saw the pro-caspase-8 and pro-caspase-3 from the TRAIL and gemcitabine group. In the combination group, we can not only see the pro-caspase-8 and pro-caspase-3, but also their cleavages, this study indicated that the combination of gemcitabine and TRAIL have synergistic apoptosis effects. The combination group disarmed the Caspase-8 inhibition of state, through some mechanism, and restored apoptosis signals, promote cell apoptosis occurs. The inhibition of Caspase-8 state, restored apoptosis signals, promote cell apoptosis occurs.We continue our application by using Western blot methods to test the DR4, DR5 expression of combination group, also examined the upstream adjust Caspase-8 Flip expression protein. The results showed that before and after gemcitabine group treatment, DR4 and DR5 expression didn't happen obvious changes, but c-Flip expression was obviously cut down. So explain the mechanism of gemcitabine improving the sensitivity HCCC-9810 to TRAIL is not dependent on the DR4, DR5 express hike, but because of the downgraded c-Flip express, removing the inhibition of Caspase-8, lead apoptosis.Based on our experimental results, we can draw following conclusions:(1) Small does TRAIL has little effect on HCCC-9810,and perform drug tolerance to TRAIL. (2) Gemcitabine has effection to HCCC-9810 rely on both time and drug concentration.(3) Gemcitabine can reinforce the sensitivity to TRAIL in HCCC-9810 cells. (4)TRAIL combined with gemcitabine coused HCCC-9810 cell line death by apoptosis.(5) TRAIL and gemcitabine inducing apoptosis of HCCC-9810 have significant synergies. The mechanism may release the inhibition of caspase-8 by down-regulation of the expression of c-Flip and then resume apoptosis signaling transduction. there is no relation with DR4 and DR5.