Mitochondria encoded Cytochrome B (CYTB) mutations were reported in different cancers; however, the impact of these mutations in cellular metabolism and growth is unknown. In the present study, we overexpressed a 21 base pair deletion mutation (mt) and the wild type (wt) CYTB in a murine bladder cancer cell line MB49 which closely resemble human bladder cancer. Following stable transfection, we examined in vitro and in vivo tumor growth, distribution of mitochondria, production of reactive oxygen species (ROS), lactate, ATP and oxygen consumption. We examined the expression of apoptosis, cell cycle and metastasis regulatory proteins including Bcl-2, Bcl-x/L, Bad, Bax, Cytochrome C, lamin B1, Akt, p-Akt, NFκB2, CyclinD1, Cdk4/6 and MMP-2 in the CYTB overexpressing cells. The mt and to a lesser extent wtCYTB overexpressioninduced significant in vitro and in vivo tumor growth. Both mt and wt CYTB transfected cells produced significant amount of ROS, exhibited elevated expression of NFκB2, CyclinD1, Cdk4/6 and MMP-2 and were significantly resistant to apoptosis. Increased copies of structurally abnormal mitochondria with highly polarized membrane potential, increased nuclear lamin B1 and higher expression ratio of Bcl-2/Bcl-xL:Bax/Bad were evident in the mt as well the wt CYTB overexpressing cells. The mt and wtCYTB overexpressingcells also exhibited significant invasive phenotype, increased mean vessel density (MVD) and expression of VGFR-2 accompanied by decreased oxygen consumption and higher lactate production. Inhibition of NFκB2 activation induced apoptosis with increased Bax:Bcl-2 ratio, decreased CyclinD1, cleaved PARP and release of Cytochrome C in the cytosol. Inhibition of ROS production by Vitamin-E treatment also significantly inhibited proliferation of the wt and mt CYTB transfected cells. Moreover, mt and wt tumorderived ROS induced significant in vitro lysis of normal splenocytes. Thus, overexpression of both mt and wt CYTB resulted in significant tumor growth by triggering rapid cell cycle progression and inhibiting apoptosis through upregulation of NFκB2 signaling pathway and tumor-derived ROS was utilized for killing immune cells as a mechanism of immune evasion. Similar to MB49 cells, we observed significant in vitro proliferation, colony formation; increased number of structurally abnormal mitochondria and ROS production in SV40 transformed human uroepithelial SV-HUC-1 cells overexpressing wt and mt CYTB. The CYTB overexpressing SV-HUC-1 cells also exhibited low oxygen consumption, lower ATP and higher lactate production. Thus, overexpression of mt and wt CYTB appeared to have triggered similar pathways of cell proliferation in mouse and human uroepithelial cells. These observations may have future therapeutic application by using agents including antioxidants, NFκB inhibitors to selectively kill tumor cells with increased number of mutant mitochondria.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA