| Backgrounds and Objectives1. Recombinant oncolytic adenovirus (OAds) refers to a class of genetically modified adenovirus which can replicate in tumor cells, damage the host cells, and continue to damage cells through the release of progeny virus. One paper published on Science has reported that oncolytic virus Onyx-015 can only replicate in tumor cells with mutant p53 and kill the cells, while shows no effects on normal cells, in which the gene encoding E1B55KD was knocked out. This is an important milestone for OAds treatment for tumors. However, the oncolytic effect of OAds itself is far from the needs for tumor treatment, so OAds are transformed into vectors containing multiple cloning sites, thus anticancer genes can be inserted into vectors and proliferate along with OAds. Massive replication of OAds in tumor cells can amplify the anticancer gene carried by the virus, thus improving the duration of its expression and increasing the diffusion capacity in tumor tissue.Recent studies reveal various biological activities of IL-24, including inhibition of tumor cell growth, induction of tumor cell apoptosis, inhibition of angiogenesis, and enhancing the sensitivity of tumor cells to chemotherapy and radiotherapy to some content. Ki67, a cell proliferation-associated antigen closely associated with cell proliferation and prognosis of the tumor, has become a most commonly used pathological marker for clinical determination of tumor grade and prognosis in tumors, such as melanoma. Recently, we cloned a strong tumor specific promoter, Ki67 promoter (Ki67-promoter). Strong transcriptional activity of Ki67-promoter can be observed in 13 strains tumor cell lines from 5 species, including melanoma. Ki67-promoter showed very high transcription activity and tumor specificity. Meanwhile, it is only 253bp in size, making it easy to be inserted into virus vectors, and be used to control the highly effective specific expression in gene therapy. In the present study, a recombinant OAds Ki67-ZD55-IL-24 carrying the therapeutic gene IL-24 under the control of Ki67 promoter was constructed. The effects of this OAds on cell proliferation and apoptosis in melanoma A375 and M14 cells and normal lung embryo fibroblast NHLF were studied.2. Curcumin, an extract from turmeric and a natural polyphenol, functioning as free radicals scavenger, antioxidant, and antitumor, anti-inflammatory, and antiviral agent, as well as others, has been widely used as an anti-inflammatory and lipid-lowering drug. The antitumor effect of curcumin and the mechanism underlying are now the hotspots for researchers worldwide. Many studies showed that curcumin can inhibit the growth of various kinds of tumors, such as liver cancer, head and neck squamous cell carcinoma, and colon cancer. It is classified as the third generation of cancer chemo-preventive agent by American National Cancer Institute and a new anticancer drug in the 21st century by the FDA. The anticancer mechanism underlying is complicated, involving different signaling pathways in various cell types, including the down-regulation of AP-1, Egr-1, uPA, COX2, NOS, TNF, cell surface adhesion molecules and cyclin D1, and the inhibition of JNK, protein tyrosine kinase, and protein serine kinase/threonine kinase, without any obvious side effects. Although curcumin has been demonstrated to have anti-cancer effect in various tumors, its anticancer effect on melanoma cells and underlying mechanism are still unclear.In the present study, the effect of curcumin on the growth of melanoma cells was determined. Protein expression of NF-κB and MAPK pathways-related proteins, and mitochondrial apoptosis and death receptor pathways-related proteins in melanoma cells treated with different concentrations of curcuminwere detected semi-quantitatively using Western blotting and reverse transcription enzyme-linked immunosorbent assay. The potential and related mechanism for curcumin involved in anti-tumor effect against malignant melanoma were explored.Methods and results1. The anti-melanoma activity of four kinds of OAds (i.e. ZD55-EGFP, Ki67-ZD55, ZD55-IL-24, and Ki67-ZD55-IL-24) was investigated first in vitro. Melanoma cells A375 and M14, and normal lung fibroblast NHLF cells were treated with four kinds of OAds respectively. The repilcaiton of OAds was confirmed by detecting the expression of early viral protein E1 A using Western blotting. The expression of IL-24 was determined using RT-PCR, Western blotting, and ELISA. The results showed that Ki67-ZD55-IL-24 can be replicated and proliferated in A375 and M14 melanoma cells with high expression level of IL-24. Crystal violet staining was used to examine the cytotoxicity of the OAds, revealing a stronger cytotoxicity of Ki67-ZD55-IL-24 than that of ZD55-EGFP, ZD55-IL-24, and Ki67-ZD55. Cell survival rate calculated by MTT showed distinct inhibition effect of Ki67-ZD55-IL-24 on the proliferation of melanoma A375 and M14 cells, which was more significant than that of Ki67-ZD55-IL-24 and ZD55-IL-24 (P<0.05). However, no obvious inhibition effect on normal lung fibroblast NHLF was observed. Apoptosis index and cell cycle were detected by flow cytometry (FCM). The data showed that Ki67-ZD55-IL-24 can induce the apoptosis of melanoma cells with a much higher induction rate than that of ZD55-EGFP, Ki67-ZD55, and ZD55-IL-24 (P<0.05). Compared with the control, Ki67-ZD55-IL-24 can obviously arrest A375 cells at G2/M phase (43.10%). Cell migration and invasion capacities of melanoma cells detected by cell migration and invasion assays indicated that the migration capacity of A375 and M14 cells treated with Ki67-ZD55-IL-24 was reduced by 81% and 84%, respectively, and the invasion ability was decreased by 82% and 79%, respectively. The expression profile of apoptosis-related proteins Bax, Bcl-xL, Mcl-1, and Caspase-3 detected by Western blotting showed significantly increased levels of E1A and Bax, decreased expression levels of both Bcl-xL and Mcl-1, and distinct protein splicing in Caspase-3 in melanoma cells infected with Ki67-ZD55-IL-24.Next the anti-melanoma activity of OAds was investigated in vivo using mouse model. Transplantation tumor model of malignant melanoma was established in nude mice. The volume of tumor, the weight of mice and the average weight of the tumor in nude mice were measured with the intervention of ZD55-EGFP, Ki67-ZD55, ZD55-IL-24 and Ki67-ZD55-IL-24, respectively. Data showed that the average tumor volume in mice treated with Ki67-ZD55-IL-24 was 1287.8±290.12mm3 30 days after intervention, with statistical intergroup differences (P<0.05). Thirty days after intervention, the average tumor weight was 1.15±0.31 g in Ki67-ZD55-IL-24 treated mice, with statistical intergroup differences (P<0.05). No significant differences were found in the weight of mice among all groups 30 days after intervention. E1A expression was tested using Western blotting, and it was shown that E1A can be efficiently expressed n melanoma treated with Ki67-ZD55-IL-24. Immunohistochemistry was used to detect the expression of IL-24 protein, showing that IL-24 was replicated and expressed in melanoma xenograft in nude mice. Detection of tumor tissue necrosis by HE showed necrosis and the cell nuclear hyperchromatic, pyknosis, fragmentation, and fibrosis in large areas in Ki67-ZD55-IL-24 group. The apoptosis of tumor tissue was measured by TUNEL and the results showed that the apoptosis index of Ki67-ZD55-IL-24 treatment (71.62 ± 7.31%), was statistically different from that of PBS (12.32±2.84%), ZD55-EGFP (28.01±1.82%), Ki67-ZD55 (32.66±3.12%), and ZD55-IL-24 treatment (46.25± 7.07%) (P<0.05), respectively.2. Cell apoptosis in human melanoma cell lines A375, MV3, and M14 treated with curcumin was measured using morphological method, Hoechst 33258, Annexin V/PI 33258 fluorescence staining and FCM, and the data showed that curcumin can significantly inhibit the growth of human melanoma cells, and induce tumor cell apoptosis, manifesting as cell shrinkage and nuclear chromatin condensation; The apoptosis index tended to increase with the concentration of curcumin. Western blotting assay was used to detect the expression ofapoptosis-related proteins P53, p38, Bcl-2, γ-H2AX, Caspase-3, Caspase-8, NF-κB, Bax, and Mcl-1. The results showed that during the induction of apoptosis of human melanoma cells by curcumin, compared with that before treatment, the expression levels of apoptosis-related proteins γ-H2AX, p53, and t-Bax were increased and the expression levels of p38, Bcl-2, Caspase-3, Caspase-8, NF-κB, Bax, and Mcl-1 were decreased.ConclusionRecombinant oncolytic adenovirus carrying Ki67 promoter and therapeutic gene IL-24 can significantly inhibit the growth of both melanoma cells and melanoma xenograft in nude mice, in which the inhibition degree by Ki67-ZD55-IL-24 is higher than that of ZD55-EGFP, Ki67-ZD55, and ZD55-IL-24, and the apoptosis-promoting effect is more distinct. The mechanism of cell proliferation and apoptosis caused by curcumin in melanoma cells may be related to the regulation of apoptosis-related proteins γ-H2AX, p53, t-Bax, p38, Bcl-2, Caspase-3, Caspase-8, NF-κB, Bax, and Mol.1... |
