BVAN08 Induces Mitotic Catastrophe And Radiosensitization Of Liver Cancer Cells In Vitro And In Vivo

Cell death is an important physiological and pathological phenomenon, which is also the fundamental basis of cellular response to cancer therapy. Cell death can be mechanistically and morphologically categorized into apoptosis, necrosis, autophagy and mitotic catastrophe. Mitotic catastrophe occurs in mitotic cells as the consequence of abnormal cell division, it is often accompanied by abnormal cell cycle checkpoint or the destroyed spindle structure. Mitotic catastrophe has been taken as a new strategy for the development of new anticancer drugs, especially against the cancers with the resistant to traditional chemotherapy treatment.DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, inincluding another two regulatory components, i.e, Ku70 and Ku80. DNA-PKcs was firstly identified in the pathways of the nonhomolgous end joining (NHEJ) of DNA double-strand breaks and V(D)J recombination. In addition, DNA-PKcs is important for maintaining telomere length and stability, and it can phosphorylate a lot of essential proteins in vitro and in vivo, such as oncogenes products and signal pathway factors (e.g.c-fos, c-myc, oct-1, c-jun), DNA damage response proteins (e.g. H2AX,p53,Chk2,Artemis,XRCC4) and so on.Lately, overexpression of DNA-PKcs has been reported in a extensive variety of human tumour. The immunohistochemistry of liver tumour indicated that the DNA-PKcs level was higher than normal cells. Our laboratory work has found an effective inhibitor of DNA-PKcs by the vanillin derivative—BVAN08, which shows a killing effect on various cancer cells, and has the potential of development for a new anticancer drug. In order to confirm the anticancer target potential of DNA-PKcs and develop BVAN08 as a new anticancer drugs, we have made the following major experimental research achievements:(1) MTT assay, Typan blue staining, Annexin V/PI staining and fluorescent staining were used to analyze the effect of BVAN08 on cell proliferation and apoptosis of HepG2 cells, single cell gel electrophoresis and pulsed-field gel electrophoresis were used to analyze DNA damage and repair. The results indicated that BVAN08 exhibited an effective antiproliferation against HepG2 cells, induced apoptosis and DNA double strand break. On the base of observation of G2/M arrest induced by BVAN08, and the mitotic arrest, spindle structure destruction (aberrant multi-polar spindles) and multinucleated cells were further demonstrated by laser confocal microscopic observation of BVAN08-treated cells after immunostained with the anti-γ-tubulin antibody and anti-?-tubulin antibody. Western blot analysis indicated that cell cycle control associated transcription regulation factor FoxM1 and DNA-PKcs were down-regulated, while the checkpoint kinase Chk2 was activated by BVAN08. Our results indicated that BVAN08 induced mitotic catasphophe of HepG2 cells. Inactivation of FoxM1and DNA-PKcs proteins could involve in the mechanistic pathway of mitotic catastrophe induced by BVAN08.(2) In order to further reveal the signal pathway which is responsible for the induction of mitotic catastrophe by BVAN08, and to study the relationship between mitotic catastrophe and apoptosis, we employed the gene chip technology and proteomics technologies to investigate the mechanism of mitotic catastrophe. The results showed that CDKN1A, CCNE1, CDKN1C, CDKN2D were upregulated and CDC2 was decreased in HepG2 cells treated with BVAN08. The proteomic analyses have identified 93 differentially expressed protein spots which matched 137 peptides, including these 45 increased protein spot(matching 76 peptides), and 48 protein decreased spots(matching 61 peptides). These results suggested that a number of cell cycle-related genes participate in mitotic catastrophe, 14-3-3 signal pathway, and PI3K/AKT signal pathway involve in mitotic catastrophe.(3) In order to further investigate the function of DNA-PKcs in mitotic catastrophe, RNA interference technique was used to decrease the protein level of DNA-PKcs. Stable HepG2 cells line which transfected with DNA-PKcs siRNA vector were selected. In order to compare effect of three anticancer drugs (DRB, DDP, VP-16) on the growth inhibition of DNA-PKcs-depleted HepG2-H1 cells. The results showed that these anticancer drugs inhibited the growth of HepG2-H1 cells and significantly disturbed the cell cycle progression. For example, DRB induces G2-M arrest, while VP-16 induces S phase arrest. However, DNA-PKcs depletion augments the induction of S phase arrest by DDP, and could promote the entrance of the VP-16-arrested S phase cells into G2 phase. The three drugs induced abnormal mitosis phenomenon in BVAN08 treated HepG2-H1 cells, indicated the function of DNA-PKcs in regulating the process of cell mitosis, mitotic catastrophe induced by BVAN08 was related to inactivation of DNA-PKcs protein.(4) In order to investigate the anticancer activity in vivo, HepG2 cell line expressing green fluorescent protein were established for the in vivo tumor growth monitoring of molecular image system. The results demonstrated that BVAN08 has antitumor activity on tumor-bearing mice, and no obvious side effect to body weight and peripheral white blood. Immunohistochemistry demonstrated that the expression of DNA-PKcs protein was decreased in BVAN08 treated group. These results indicated that DNA-PKcs may play a role in inhibiting tumor growth.(5) The sensitization of BVAN08 on radiotherapy of tumors has been further investigated. The results showed that combined treatment BVAN08 andγ-rays significantly decreased the cancer cells survival, increased radiosensitivity of cancer cells. Combined treatment of BVAN08 and andγ-rays remarkably inhibited the growth of tumor in vivo. The results indicated that BVAN08 has radiosensitization effect both in vivo and in vitro. Moreover, BVAN08 has less toxic to the normal tissues.(6) To study the mechanism of BVAN08 on radiosensitization and the expression of a number of critical DNA damage repair proteins, including DNA-PKcs, ATM, checkpoint kinase2 (Chk2) and Plk1 proteins. The results showed that BAN08 inhibited DNA double-strand break repair after irradiation, decreased expression of DNA-PKcs protein, while activated ATM, Chk2 sigaling pathway to increase cell radiosensitivity.This research provided the broad evidence showed that BVAN08 can induce cancer cell death in term of mitotic catastrophe. BVAN08 inhibited tumor growth in vivo, enhance the sensitivity of tumors to radiotherapy but less toxicity to normal tissues. DNA-PKcs can be used as an effective molecular target for cancer therapy and BVAN08 has the value for cancer developing as an effective anticancer drug.