Growth Inhibitory Effects And Mechanism Of ATRA Combined With IFN-α And Rh₂ On Hormone Independent Prostate Cancer Cells

Prostate cancer (PCA) is the second most frequently diagnosednon-cutaneous malignancy in American males in present, For the mosteffective therapy with the fewest side-effects, a thoroughunderstanding of the genes involved in the neoplastic process isessential. Androgens are known to play a critical role in thetumorigenic process, with activity mediated by the androgenreceptor. Initially, prostate cancers are androgen-sensitive(that is, they cease growing when deprived of androgens or treatedwith androgen receptor antagonists, such as flutamide orbicalutamide).Therefore most patients respond to androgen ablationtherapy. However, there are side-effects to this therapy that makeit unpleasant for the patient. Even with androgen ablation therapy,the disease often recurs and when it does, it usually becomesandrogen-insensitive or hormone-refractory. with the diet changingand approaching of population aging, prostatic morbility in ourcoutry have raised up year by year. In recent years, thechemotherapeurapy of prostate cancer attract the attention of mostpeople,especially drug combination often receive a comfortbalresult. Thus, our research is to study the inhibitory effects ofacid retinoid combining with interferon-αand Ginsenoside Rh2 onhuman prostate cancer cell lines PC-3,PC-3M (androgen independentcell lines) respectively and also research the expression effectof GRIM-19 (an apoptosis related gene) and STAT3 (an oncogene),providing theory clue for androgen independent prostate cancer. Themain body of this article composed two parts. The first part: It has been provided retinoic acid compounds have many effectsin inducing differentiation, inhibiting growth, inducing apoptosisto human malignant tumor etc. but its clinical result of treatmentis not very satisfied. Taking longer engender some toxicities andside effects. However, retinoid acid combind with interferon obtainsatisfying outcome in antitumor through much signal transfer waysto induce cell apoptosis. These data suggest there are stimulatedpathways existence of cross-talk between RA and IFN. Althoughthese ligands exert their effects via disparate signalingmechanisms. In 2000, JonE?Angell and his colleagues employed anantisense knock-out technique. This approach permits the isolationof cell death-associated genes GRIM-19, a novel GRIM gene, whoseexpression is induced by the RA/IFN combination. Over expressionof GRIM-19 enhances cell death in response to RA/IFN. STAT3 (signaltransducers and activator of transcription) has been suggested asan oncogene and GRIM-19 interacts specifically with thetranscription factor STAT3 and inhibits STAT3-dependent geneexpression. GRIM-19 may prove to be a novel antioncogenic protein.Method: PC-3 cell line was treated by retinoid acid or interferon-α,or in combination in vitro. Cell proliferation was evaluated byMTT assay aimed at screening the rightest medical density;Observing the cell changes by phase-contrast microscope ; Detectingapoptosis rate by AO assay; the level of GRIM-19 gene and proteinof PC-3 cells was measured by immunocytochemistry and RT-PCRrespectively .After treated with ATRA/IFN, the changes of GRIM-19and STAT3 proteins were studied by Western-blot assay.Results and Disscussion: MTT assay shows proliferation of PC-3 cells occurred aftervarious dose of ATRA/IFN-αcombination or solo application in 72hours. inhibitory rate of ATRA/IFN-αcombination has remarkabledifference compared with ATRA,IFN-α,control group (P<0.05). wescreened IFN-α1000u/ml combined with ATRA1uM group as well as IFN-α1000u/ml and ATRA1uM as followed experiment dose. we observed byphase-contrast microscope that the cell numbers decreased are morethan ones control's group. Apoptosis cells which are shrink andfloating are observed with the extension of time. According to thescreening results of MTT assay, morphors of PC-3 cells were abnormaland changed into salmon pink with AO assay after PC-3 cells weretreated with ATRA/IFN combination.Orange spot appear in PC-3 cellsindicatting cell apoptosis. Cells treated with ATRA and IFN alonedidn't appear apoptosis phenomenon and control group assume towell-distributed green color with normal shape.The consequences ofRT-PCR and immunocytochemistry demonstrated the increasing ofGRIM-19 expression treated ATRA/IFN compared with control group.STAT3 protein that treatmed ATRA /IFN were decreased significantlycompared with control and IFN-α, ATRA alone.Conclusion: Retinoid acid and interferon-αcombination can suppress thegrowth and also induce apoptosis of PC-3 cells mainly viaupregulation of GRIM-19 gene expression. Down-regulation of STAT3maybe was the results of GRIM-19 overexpression.The second part: Rh2 is a ginsenoside extracted from ginseng that has drawnattention in a few laboratories in Asian countries because of itspotential tumor-inhibitory effect. In the present study, Rh2 havea significant influence on many tumor-cell lines for its effectson cell proliferation, induction of apoptosis, and potentialinteraction with conventional chemotherapy agents. Rh2 inhibitedcell growth by G1 arrest at low concentrations and induced apoptosisAt high concentrations in a variety of tumor-cell lines, possiblythrough activation of caspases. Most interestingly, Rh2 can acteither additively or synergistically with chemotherapy drugs oncancercells.Particularly,it hypersensitized multidrug-resistant breastcancer cells to paclitaxel. These results suggest that Rh2 possessesstrong tumor-inhibiting properties, and potentially can be used intreatments for multidrug-resistant cancers.Method: 1) AO assay detecting apoptosis of PC-3M cells after treated with Rh2 for 24 hour; 2) RT-PCR measured the level of GRIM-19 mRNA in PC-3M cells treated with Rh2 for 24 hour;...