Role of Normoxic Expression of Hypoxia Inducible Factor 1-Α and Endoplasmic Reticulum Stress Associated Protein Grp-78 in the Modulation of Drug-And Radioresistance Acquisition of Human Hepatoma Cell Lines

An accumulating body of evidence has demonstrated that hypoxia is known to induce hypoxia-inducible factor 1 (HIF-1α). However, we have recently shown that one of the five hepatoma (HCC) cell lines, J5 subline, can uniquely express HIF-1αunder normoxic condition. In parallel with this interesting finding, we also demonstrated that J5 subline could express endoplasmic reticulum (ER) chaperone protein GRP-78 under a non-ER stress stimulatory situation. This unique phenomenon implies that HIF-1αand GRP-78 may be interrelated. And the mechanism(s) underlied these interesting findings remain elusive. Although the involvement of HIF-1αexpression has been implicated to be responsible for the acquisition of chemoresistance and radioresistance, yet, the experimental designs have all been conducted under the hypoxia conditions. Thus far, the comparison of HIF-1αand GRP-78 expressions with or without hypoxic conditions and their subsequent effects on the modulation of chemoresistance and RT/ PDT resistance have seldom been investigated. With the availability of J5 cells, we will be able to perform these studies with ease. For these reasons, we formulate this three-year research proposal for the purpose of unveiling the possible role of normoxically expressed HIF-1αand ER chaperone protein GRP-78 under a non-ER stimulatory condition. The year-by-year projects and their strategies are outlined below: First year: To elucidate the ubderlying mechanism(s) associated with the normoxic expression of HIF-1αand GRP-78 in J5 cells. Specifically, are the downregulation of prolyl hydroxylase (PHDs) activities involved which resulting in the failure the hydroxylation of HIF-1αor the activation of HIF-1αvia promoting PI3K/AKT signaling pathway via phosphorylation of Akt ? Second year: To dissect the roles of HIF-1αand GRP-78 in the modulation of chemoresistance of HCC cells via siRNA technique or target inhibitions of HIF-1α(inhibitors such as YC-1 and Taxol) and GRP-78 [(EGCG) and Neringenin (NGEN)]. A combined strategy using EGCG and Hsp90 inhibitor (e.g., 17-DMAG) will be evaluated for its efficacy against J5 cell carrying noroximally expressed HIF-1 and GRP-78. Third year: To dissect the role of HIF-1αand GRP-78 in the modulation of radioresistance and phototherapy resistance acquisition of HCC cells via siRNA technique or target inhibitions of HIF-1α-1 and Taxol) and GRP-78 (EGCG). Also, the role of constitutive expression of bFGF and interferone-α(INF-α)-stimulatory increases in bFGF levels in the modulation of resistances against RT/ PDT will also be investigated. This test may constitute a novel biomarker for assessing the possibility that a cancer cell will acquire RT/ PDT resistance and its clinical applicability may be enormous. In sum, all the projects outlined in this research proposal are novel and interesting. The information obtained can have tremendous clinical applicability because it can provide the basis for designing effective strategies for overcoming the chemoresistance and radioresistance acquisition of HCC cells attributtable to normoxic expression of HIF-1αand GRP-78. Further, by demonstrating HIF-1α and GRP-78 can confer chemoresistance and radioresistance acquisition will encourage scientists to actively pursuing and identifying of target inhibitors from natural sources, such as EGCG [polyphenol of tea or aspirin (sulicylate)] which have been shown to inhibit GRP-78. Active screening of natural small molecules that are capable of serving inhibitors for these two effectors will enormously benefit the physcians in treating patients haboring chemo- and radiotherapies resistance acquisition. Finally, it should be reiterated that all the proposed studies listed year by year are not mutually exclusive and so, they can be carried out simultaneously.