Hepatocellular carcinoma is the leading cause of cancer mortality in Asia. Available cytotoxic modalities used to treat the disseminated forms of this neoplasm have yielded less than optimal results. By a noncytotoxic mechanism, phorbol esters potently inhibit the in vitro growth of hepatocellular carcinoma and other neoplastic cell lines. The ability of these agents to inhibit hepatoma growth could be utilized in designing novel treatments for this neoplasia. Preliminary studies using other growth inhibitory/differentiation agents to treat leukemias and carcinomas of the head and neck have demonstrated promising results. Mitogenic effects in other tissues preclude the use of phorbol esters in vivo. Phorbol esters (TPA) exert their effects by activating members of the protein kinase C (PKC) gene family. Because of differences among the eight PKC isoforms, it is hypothesized that activation of individual isoforms may selectively mediate TPA-induced events. Thus, studies are warranted to directly assess the involvement of individual isoforms in mediating the growth inhibitory effects of TPA. In this FIRCA proposal we wish to initiate a collaborative venture between my laboratory and that of Dr. Anna Farago, Semmelweis University, Budapest, Hungary. The goal of the FIRCA proposal is to examine the ability of individual isoforms to modulate TPA-induced inhibition and directly inhibit proliferation of the human hepatoma HepG2 cell line. The experience of Dr. Farago in the PKC field compliments our own and her work in heoatocytes will greatly facilitate completion of this proposal. By examining the role of individual PKC isoforms on growth inhibition, the FIRCA proposal is a direct extension of a specific aim in the parent R01 award which examines the function of individual isoforms on growth and differentiation in the human monoblastoid U937 cell. Data obtained from the FIRCA study will allow us to assess whether growth inhibition elicited by activation of an individual isoform is a general response seen in cells of diverse lineages (e.g. hematopoietic and hepatic) or is strictly a cell-specific phenomenon. Acquisition of this information is essential to further understand the role of the PKC signal transduction pathway in modulating cellular growth and differentiation. To pursue the goal of determining the PKC isoform(s) responsible for mediating TPA-induced growth inhibition of HepG2 cells, we will: i) examine the effects of stably over expressing PKC isoforms from the group A (alpha) and B (epsilon) subfamilies on growth and DNA synthesis of the TPA-treated HepG2 cells; 2) determine whether other isoforms within their respective subfamilies can mimic this effect; 3) analyze the relative contributions of the regulatory and catalytic domains of PKC in modulating TPA-induced alterations in HepG2 growth and 4) directly determine if constitutively activated mutant alpha and epsilon isoforms can inhibit HepG2 growth in the absence of TPA. Knowledge gained from studies outlined in this FIRCA proposal could have direct clinical implications in developing modalities to treat hepatic cancer and neoplasia in general. These results would focus the development of agonists selectively activating the involved isoform and could form the basis for designing PKC mutants which could be used as a selective form of gene therapy for hepatocellular carcinomas and other neoplasms.
