Study On The Antitumoral Efficacy And Mechanism Of Adenovirus Mediated HCCS1 Overexpression

Hepatocellular carcinoma suppressor 1(HCCS1) has been identified as a novel tumor suppressor gene candidate by screening the high-frequency loss of heterozygosity(LOH) on chromosome 17p13.3 in HCC. It was shown that HCCS1 had a high frequency of mutation in one allele and loss of the other allele in HCC samples, and the protein expression level of HCCS1 reduced in cancer cells. After transfection into human hepatocarcinoma cells, HCCS1 could significantly inhibit tumor cell proliferation both in vitro and in vivo. The purpose of this study is to further confirm the antitumoral efficacy of HCCS1 and evaluate the potent of HCCS1 as a therapeutic gene for cancer therapy. On the base of the observation that the overexpression of HCCS1 could induce cell apoptosis, we sought to investigate the mechanism of HCCS1-induced apoptosis.The HCCS1 armed adenovirus, Ad-HCCS1, was constructed by using AdEasy vector system. The overexpression of HCCS1 mediated by Ad-HCCS1 was demonstrated by using ssmi-quantitatice RT-PCR and western blot assay. The recombinant adenovirus was amplified by infecting 293 cells and purified by cesium chloride density gradient ultracentrifugation. The preparation of recombinant adenovirus was titrated by a plaque assay using 293 cells. We evaluated the antitumoral effect of Ad-HCCS1 by cell viability assay in vivo. It was shown that Ad-HCCS1 could reduce the growths of both human hepatocarcinoma cells and human colorectal carcinoma cells in vivo (p＜0.01). The therapeutin effect of it was evaluated in nude mice by intratumoral injection of the recombinant adenovirus and Ad-HCCS1 was shown to inhibit the growths of human cancer xnografts significantly (p＜0.01). Meanwhile, we discovered that the increased expression of HCCS1 could induce cell apoptosis by Hoechst 33258 staining for nuclear morphological changes, TUNEL assay and Annexin V binding assay. The Bcl-2 family member Bax, but not Bid, was cleaved and inserted into mitochrondrial membrane during the HCCS1-induced cell apoptosis. The mitochondrial membrane potentials of the cells infected with Ad-HCCS1 were then collapsed and cytochrome C was released from mitochondria, which led to the activation of caspase-9 and caspase-3. But the cleavage of caspase-8 and caspase-12 were not detected during the HCCS1-induced cell apoptosis. Although AIF was also released from mitochondria together with eytochrome C, the nuclear translocation of it was not detected. In addition, it was shown that the overexpression of HCCS1 could induce the release of the lysosomal proteinase cathepsin D into cytosol. The cathepsin D inhibitor could effectively prevent the cell apoptosis caused by HCCS1 overexpression.Therefore, Our data support the role of HCCS1 as a novel tumor suppressor gene and suggest that HCCS1 may provide a new promising therapeutic gene candidate for treatment of human cancers. It is indicated that the inhibitory effect of HCCS1 on tumor cell growth is partially due to the induction of apoptosis. We further investigated the mechanism of HCCS1-induced cell apoptosis, and the results suggest that mitochondria/caspase-3/caspase-9 pathway may play an important role in cell apoptosis caused by HCCS1 overexpression. It is also indicated that increased expression of HCCS1 may trigger the lysosomal cell apoptosis pathway mediated by cathepsin D. Prohibitin(PHB) was identified a tumor suppressor gene candidate. PHB acts as a molecular chaperone when it locates in the inner membrance of mitochondria and acts as a TF when in the cell nuclear. Recently, its expression has been shown to up-regulated in a variety of tumor tissues and in the surem of cancer-beating patients. The purpose of the present study is to investigate the PHB expression levels in the tumor tissues and the serum from native gastric and colorectal cancer patients, and evaluate the potent of serum PHB levels as a biomarker for gastric and colorectal cancers.The PHB gene was cloned into the prokaryotic expression vector pQE30 to construct pQE30-PHB. The recombinant protein was expressed by IPTG induction and purified through a Ni-NAT resin column. The anti-PHB monoclonal antibody was obtained from a hybridoma produced by fusing spleen cells from the BALB/c mice immunized against the purified PHB protein with SP 2/0 mouse myeloma, and purified from mouse ascetic fluid by protein A Sepharose affinity chromatography. One strain anti-PHB monoclonal antibody with high affinity was obtained by ELISA detection and Western blot assay. The PHB protein expressions in tumors were measured by immuno-histochemical staining, and it was demonstrated that its expression in gastric and colorectal cancer tissues was up-regulated compare to the matched noncancerous tissues (p＜0.01), and PHB expression level was correlated with the degree of tumor histological differentiation. The poorly differentiated tumors expressed more PHB proteins (p＜0.01). On the base of this, we established the time-resolved fluoroimmunoassay(TRFIA) of PHB protein and detected the serum PHB level of gastric and colorectal cancer patients by using it. However, there was no difference between the serum PHB levels from gastric or colorectal cancer patients and matched normal volunteers(p＞0.05). We subsequently proved that gender or age of patients was not the bias which might affect the result in this study, although that there was significant difference between the serum PHB levels of the male and female normal volunteers.Our results indicated that PHB is a potential tissue biomarker for local gastric or colorectal cancer. Histology-based measure of PHB protein is an assistant tool to diagnosis, cure and prognosis for gastric and colorectal cancers. But serum PHB as a biomarker for cancers is very debatable.