Establishment Of Transmitochondrial Cell Model For MtDNA Depleted SK-Hep1 Cells And It's Biological Characteristics Analysis

The mechanism of tumorigenesis remains unclear. Mitochondrion is the most capital organelle, and its dysfunction is one of the most profound features of cancer cells. Mitochondria are dynamic organelles that play a central role in cellular metabolism, and mitochondria are also sites for several additional key cellular processes such as the oxidative decarboxylation of pyruvate, the tricarboxylic acid cycle, fatty acid oxidation, cellular movement, cellular proliferation and apoptosis. Mitochondria are involved either directly or indirectly in many aspects of altered metabolism in cancer cells, and several notable differences have been discovered between the mitochondria of normal cells and that of transformed cells. For example, various tumor cells exhibit differences in the number, size and shape of their mitochondria relative to normal controls. The mitochondria of rapidly growing tumors tend to be fewer in number, smaller and have fewer cristae than mitochondria from slowly growing tumors; the latter are larger and have characteristics more closely resembling those of normal cells.MDR, the major cause of Chemotherapeutic failure in cancer, is a multifactorial phenomenum. Overexpression of transporter glyco-proteins such as P-gp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 are some of the most well known MDR effectors. MtDNA deletion and mitochondrial dysfunction may be relevant to tumorogenesis, tumor metastasis and multidrug resistance. However, it is not clear whether the mtDNA-depletion is a contributing factor in the expression of molecules responsible for drug resistance. Here a human hepatoma cell lines (SK-Hep1) were used , SK-Hep1 cells were cultured in the media with EB and uridine and pyruvate for 21 generations to establish aρ0SK-Hep1 cell lines lacking mitchondrial DNA and polyethylene glycol was used as fusion promoting reagent to establish transmitochondrial cell model. Cells growth status and the ability of cell invasion were observed. Multidrug resistance was detected by MTT assay, Western blotting was applied to detect P-glycoprotein,MRP1,Bcl-2 and Bax proteins expression.We performed this study to clarify the exact mechanisms about cell's malignant phenotypes and multidrug resistance involved.Objective1. Establishment ofρ0 SK-Hep1 cells lacking mitchondrial DNA(mtDNA)2. Normal human blood platelets were used as mitochondrial donors, and polyethylene glycol was used as fusion promoting reagent to establish transmitochondrial cell model.3. To observe the biological differences between SK-Hep1,ρ0SK-Hep1 and SK-Hep1Cyb cells, including cell growth status,invasive capability, and multidrug resistance.4. Expression of P-glycoprotein,MRP1,Bcl-2 and Bax proteins were detectedMaterials and methods1. Establishment ofρ0SK-Hep1 lacking mitchondrial DNA(mtDNA)2. Human hepatocellular cancer cell line SK-Hep1 was treated by ethdium bromide (EB). The cells were cultured in media with uridine and pyruvate, and expression of COX I and COX II were detected by PCR,southern hybridization and Western blotting to verify the cells lacking mtDNA.3. Establishment ofρ0SK-Hep1 cells transmitochondrial model(Cybrids). Cybrid cells had objective fragments of mtDNA confirmed by PCR,Southern hybridization and Western blot.4. The proliferative and invasive ability of SK-Hep1,ρ0SK-Hep1 and SK-Hep1Cyb cells were determined by MTT and Transwell tests.5. Multidrug resistance was detected by MTT assay. P-glycoprotein,MRP1,Bcl-2 and Bax proteins expression was detected by Western blottingRusults1. After exposure to EB for 1 day, some SK-Hep1 cells began swelling and suspending, almost all cells died for about 10-12 days when cultured in the midia without uridine and pyruvate. When SK-Hep1 cells were cultured in the media with EB and uridine and pyruvate for 21 generations, COX I and COX II can not be amplified, and no COX I and COX II hybridization bands were found by Southern blotting, and Western blotting analysis confirmed the cells had no positive COX II protein activity.2. We fusedρ0SK-Hep1 cells with platelets to obtain SK-Hep1Cyb cells. PCR, Southern hybridization and Western blot confirmed that cybrid cells had objective fragments of mtDNA and positive COX II protein activity. SK-Hep1Cyb cells showed lower growth rates and less invasive capability than SK-Hep1 andρ0SK-Hep1 cells.3. For determining the cytotoxicity of different drugs on cells, SK-Hep1,ρ0SK-Hep1 and SK-Hep1Cyb cells were incubated with increasing concentrations of different drugs for 48 h or left untreated. When we used ADM to treat SK-Hep1,ρ0SK-Hep1 and SK-Hep1Cyb cells, the IC50 values were 0.62μg/ml,4.93μg/ml and 0.57μg/ml respectively. The RI ofρ0SK-Hep1 cells is 7.95 times more than SK-Hep1 cells, moderate drug resistance. SK-Hep1Cyb cell's 1/RI is 1.05 times more than SK-Hep1 cells, no obvious drug resistance.ρ0SK-Hep1 cells had a lower drug resistance to DDP compared with SK-Hep1 cells and a moderate drug resistance to DDP compared with SK-Hep1Cyb cells. All the cells were no obvious drug resistance to VCR and 5-FU.4. P-glycoprotein, MRP1, Bcl-2 and Bax proteins expression are up-regulated inρ0SK-Hep1 cells, and significantly increased the Bcl-2-to- Bax ratio. Interestingly, In SK-Hep1Cyb cells, MRP1 protein expression was lower and Bax protein expression was higher compared with SK-Hep1 cells, others were approximate.Conclusions1. Transmitochondrial cell model for SK-Hep1 mt-DNA depleted cells was successfully established.2. SK-Hep1Cyb cells showed lower growth rates and less invasive capability than SK-Hep1 andρ0SK-Hep1 cells.3.ρ0SK-Hep1 cells were resistance to ADM and DDP induced apoptosis than SK-Hep1 and SK-Hep1Cyb cells.4. P-glycoprotein, MRP1, Bcl-2 and Bax proteins expression were increased inρ0SK-Hep1 cells, this might be responsible for the drug resistance ofρ0 cells.