Abstract
Arsenic compounds are common, naturally occurring substances. They have been reported to be human carcinogens associated with malignancies of the lung, bladder, skin, liver, kidney, and prostate. However, arsenic compounds have been used as therapeutic agents for more than 2,400 years. Recently, arsenic trioxide (As2O3) was wildly used to cure acute promyelocytic leukemia (APL). The Food and Drug Administration (FDA) of the USA has approved arsenic trioxide for the treatment of APL in September 2000.Cervical cancer is the second leading cause of death from cancer in women worldwide. Over 90% of human cervical cancer cells are infected with different types of human papillomaviruses (HPVs) and possess wild type p53 and Rb tumor suppressor genes. But in these cells, normal p53 and Rb functions are abolished by HPVs E6 and E7 oncogenes, respectively. Therefore, restoration of p53 or Rb function by blocking E6/p53 or E7/Rb pathway might be a potential therapeutic purpose for these cancer cells. In the present study, we investigated the effects of sodium arsenite (SA) on HPVs E6 and E7 oncogenes, the effects of SA on apoptotic responses, and try to promote SA as a potential therapeutic agent for HPV-positive cancer cells.Two systems were used in this study: (1) the human lymphoblastoid cells (TK6 cells), and their E6-transfectants (TK6-E6 cells), (2) the human cervical carcinoma cells originally infected with HPV-16 (SiHa cells). Treatment with SA, but not X-ray, decreased the E6 mRNA levels and induced the expressions of p53, and its down stream genes, p21, and mdm2 in TK6-E6 cells. SA enhanced apoptotic responses, including more sub-G1 percentages, more activated caspase-3 fragments (17 kD), and more DNA ladder in TK6-E6 cells than these in their parental TK6 cells. According to the results from MTT assay, TK6-E6 cells were more sensitive to SA, but more resistant to ionizing radiation (IR) than TK6 cells. It implied that instead of IR, SA could be a potential therapeutic agent for E6-possitive cancer cells.In human cervical cancer cells, SA down-regulated E6 and E7 mRNA levels, restored p53 tumor suppressor pathway, and induced Rb expression. While SA restored normal p53 function, G2/M arrest in the cell cycle progression and apoptosis occurred. Transfection of a dominant-negative p53 markedly reduced the SA-induced apoptosis, indicating that p53 mediated SA-induced apoptosis in SiHa cells.Besides p53, other molecules are involved in regulation of apoptosis. Cyclic AMP-dependent protein kinase A (PKA) has been reported to inhibit E6 function by phosphorylation of threonine residue at its C-terminal region, which is not involved in p53 binding and degradation. But, E6 binds to Bak, an apoptosis inducer, at this region. In this study, I investigated the roles of PKA in regulation of SA-induced apoptosis. Pretreatment with a PKA inhibitor, HA1004, significantly reduced the SA-induced apoptotic cell death in E6-positive SiHa and TK6-E6 cells, but not in E6-negative TK6 cells. It implied that PKA played some roles in regulation of E6 function, which was involved in SA-induced apoptosis, in E6-positive cancer cells. Furthermore, pretreatment with HA1004 decreased SA-induced Bak expression only in E6-possitive cancer cells. However, HA1004 did not affect SA-induced p53 expression in both E6-positive and E6-negative cancer cells. These results suggested that PKA inhibited E6 degrading SA-induced Bak, and resulted in apoptotic cell death in E6-positive cancer cells, but PKA could not inhibit the E6 function of degradation of SA-induced p53.Base on these above results, SA-induced apoptotic cell death in HPV-positive cancer cells might result from the following: (1) down regulation of E6 and E7 oncogenes, (2) restoration of the p53 tumor suppressor pathway, and (3) at least in part, involvement of PKA in inhibition of Bak degradation by E6.