Combined Effect Of Curcumin With Chemotherapeutic Agents Fluorouracil On Proliferation And Apoptosis Of HepG2Cells

Objective: Hepatocellular carcinoma (HCC), a major form ofmalignancy of liver with a worldwide increasing incidence, is one of the mostcommon causes of cancer related deaths in the world. Fluorouracil is a widelyused chemotherapeutic drug for solid tumor treatment. It initiates apoptosisthrough interfering with nucleic acid synthesis, and eventually inhibits cellgrowth. However, its clinical use has been limited by its poor efficacy in HCCand wide range of side effects. Curcumin (CU), a kind of phenolic compoundfrom turmeric, has been shown to inhibit growth of several types of malignantcells both in vivo and in vitro through effects of promoting apoptosis and cellcycle arrest. Furthermore, its no or almost no toxic effects on normal cells holdclinic promise for cancer treatment. Here we investigated whether combinationtreatment of CU with Fluorouracil can significantly increase anti-tumoractivities against human hepatoma HepG2cells in vitro, while with less sideeffects. CCK-8assay and double staining flow cytometry were used to test thecell viability and apoptosis. Western Blot was then conducted to determine theapoptosis signal pathway protein (active Caspase-3) and cell survival pathwayprotein (Bcl-2). Methods: Human hepatocellular carcinoma HepG2cell inlogarithmic phase was chose and divided into CU, FU, CU+FU and negativecontrol group. After72h, CCK-8assay was used to detect the cell viability.Double staining flow cytometry was then conducted to examine apoptosis. Furthermore, apoptosis signal pathway protein (active Caspase-3) and cellsurvival pathway protein (Bcl-2) were determined by Western Blot method.Results: The results of CCK-8showed that gradual increase of drugconcentration (3.125、6.25、12.5、25、37.5、50、100μM) displayed increasedinhibition of cell viability in ether CU alone (inhibition rate is20.59±2.88%,33.53±4.63%,49.63±3.37%,64.05±3.62%,66.83±4.16%,73.08±5.29%,80.40±2.23%， respectively) or FU group (25.66±3.85%,42.15±3.58%,58.05±5.06%,65.92±4.42%,72.89±5.04%,77.14±3.93%,82.39±4.90%，respectively). Combination of CU and FU further enhance the effect of growthinhibition. FU:CU=1:2group (3.125+6.25、6.25+12.5、12.5+25、25+50、50+100μM) resulted in36.66±3.85%,63.15±2.58%,76.05±2.06%,82.92±3.41%,95.89±5.03%inhibition rate respectively. While there were30.58±2.87%,57.52±2.63%,72.62±1.36%,79.04±1.61%,93.82±2.16%inhibition rate in FU:CU=2:1group (6.25+3.125,12.5+6.25,25+12.5,50+25,100+50μM). Compared with the IC50of CU (15.32±2.60μM) and FU(11.03±1.86μM) alone, Combining CU with FU significantly increasedsensitivity of the HepG2cell to CU and FU, and the corresponding IC50for CUand FU were10.86±0.61μM and4.18±0.89μM in CU:FU=2:1group while11.53±1.73μM and8.28±1.89μM in group of CU:FU=1:2(P<0.05comparedwith alone group). Flow cytometry data revealed that the apoptotic rate in CU(25μM) and FU (12.5μM) group was7.08%and16.36%respectively, incontrast, combining CU (25μM) with FU (12.5μM) resulted in remarkable increase of apoptosis with20.43%. Such above results were further interpretedby Western Blot test. Active Caspase-3protein was significantly up-regulated inCU+FU group compared with CU or FU alone suggesting that apoptosis signalpathway was further enhanced. Meanwhile, Bcl-2protein indicating cellsurvival was obviously decreased by CU+FU treatment compared with CU orFU alone. Conclusion: The present study investigated combined effects of CUand FU on the growth of human hepatocellular carcinoma HepG2cells in vitro.The cell viability of HepG2cells was more significantly inhibited bycombination of CU and FU than CU and FU alone. This was partially explainedby higher percentage of apoptotic cells in combination group. Signal pathwayassay further showed that combining CU and FU resulted in more increasedactive-Caspase-3and decreased Bcl-2protein, causing more apoptosis and lesscell survival. Taken together, these results suggest that combining CU and FU ismore effective than CU or FU alone in vitro experiment. Furthermore,combining therapy also significantly decrease the dose of FU. Indicating thatcombined treatment of CU and FU will be a potential therapeutic methodagainst HCC with higher efficacy and lower side effects in clinic practice.