Finally, cleaved caspase-3 turned on the caspase cascade and triggered cell death. cyclin D1), leading to cell cycle arrest at the G0/G1 phase in HCC cells. BP/LPPC induced cell apoptosis through activation of both the extrinsic (Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways and activated the caspase cascade to trigger HCC cell death. In conclusion, the LPPC complex improved the antitumor activity of BP in terms of cytotoxicity, cell cycle regulation and cell apoptosis, and BP/LPPC synergistically inhibited cell growth during combination treatment with VP-16 in HCC cells. Therefore, BP/LPPC is potentially a good candidate for clinical drug development or for use as an adjuvant for clinical drugs as a combination therapy for hepatocellular carcinoma. 1. Introduction Hepatocellular carcinoma (HCC) represents the second and sixth leading cause of cancer death in men and woman worldwide, respectively. It is especially prevalent in East Asia and sub-Saharan Africa, where it is one of the leading causes of cancer-related death [1C3]. Because of ZED-1227 the long duration of HCC, most patients are diagnosed in the intermediate or advanced stages, for which chemotherapy is the only option. However, there is a low response rate and a high rate of severe side effects for chemotherapy in HCC patients [4]. Standard chemodrugs have high toxicity and lack selectivity between malignancy cells and normal cells, and it has been reported that chemodrugs accumulate in tumor tissue at 5-10% of the dose that accumulates in normal organs [5]. Therefore, the poor accumulation of chemodrugs prospects to poor prognosis, malignancy recurrence, and poor survival. It is urgent that new therapeutic options with high anticancer effects and low cytotoxicity for normal cells are developed for HCC therapy. Drug carriers, such as polymer-based liposomes, have improved the effects of drugs. These service providers protect the natural compound, decrease the drug penetration of normal organs, and increase the cytotoxicity of the drug in tumor cells [6C8]. Previous studies showed liposome-enhanced anticancer effects in colon carcinoma, osteosarcoma, pancreatic malignancy, and hepatocellular carcinoma in vitro and in vivo [9C12]. Polycationic Liposome Made up of PEI and Polyethylene Glycol Complex (LPPC), a novel modified liposome, has a lipid bilayer composed of DOPC and DLPC that is noncovalently altered with PEG and PEI [13]. The LPPC technology enhanced antitumor effects by triggering the quick penetration of drugs into tumor areas to suppress tumor ZED-1227 growth and increase drug cytotoxicity in drug-resistant malignancy [14]. Additionally, the LPPC-delivery system had improved drug transport properties and therapeutic efficacy, suggesting that it is a promising new tool for malignancy therapy [15C17]. Recent studies showed that n-butylidenephthalide (BP), a natural compound from value 0.05 was considered statistically significant. 3. Results 3.1. BP/LPPC Induced Cytotoxicity in HCC Cells Illustration of LPPC with BP is usually shown in Physique 1(a). Our previous study demonstrated that this maximal encapsulation capacity of LPPC (1?mg) was ~830? 0.05). bSignificant difference in BP/LPPC treatment compared with BP/Lipo treatment ( 0.05). cSignificant difference in BP/LPPC treatment compared with VP-16 treatment ( 0.05). 3.2. LPPC Guarded BP Activity for Cytotoxicity of HCC Cells To analyze the protection effect of LPPC encapsulation on BP activity, the drugs prepared for the BP/LPPC group (encapsulated BP) and for the BP group (nonencapsulated BP) were stored at 4C or 37C in different environments, and the drug preparations were incubated for 0, 4, 8, or 24?h. In Physique 2, BP/LPPC prepared in ddH2O answer and incubated at 4C experienced higher cytotoxicity in the two HCC cell lines (IC50 = 12.52C12.93? em /em g/ml, 0?h; IC50 = 19.17C23.83? em /em g/ml, 24?h) than the BP prepared in the same way (IC50 = 50.36C97.36? em /em g/ml, 0?h; IC50 = 243.20C275.08? em /em g/ml, 24?h). In when prepared in a protein-rich medium and incubated at 37C, BP/LPPC also showed higher cytotoxicity (IC50 = 17.41C18.68? em /em g/ml, 0?h; IC50 = 30.69C36.00? em /em g/ml, 24?h) than BP (IC50 = 47.53C95.61? em /em g/ml, 0?h; IC50 = 211.40C222.94? em /em g/ml, 24?h). The data suggest that BP was quickly losing activity after it was prepared Tgfb3 in an aqueous answer, but LPPC encapsulation guarded the cytotoxicity of BP in HCC cells. Open in a separate windows Physique 2 LPPC encapsulation guarded ZED-1227 BP activity ZED-1227 and cytotoxicity. BP/LPPC and BP stored in ddH2O at 4C or in protein-rich answer (10% FBS in PBS) at 37C for 0, 4, 8 or 24?h. (a) HepG2 and (b) J5 cells were treated with incubated-BP/LPPC or BP for 24?h, and the IC50 was calculated by using the MTT assay. ?: Significant difference compared with 0?h in the ddH2O group ( em p /em ? 0.05). #: Significant difference compared with 0?h in the protein-rich answer group ( em p /em ? 0.05). 3.3. LPPC Enhanced Cell Uptake of BP.