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Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

In prostate cancer, p53 maximizes apoptosis in response to severe DNA damage, not DNA replication stress. Here, we examined the apoptotic response of two glioblastoma cells, p53-wild type U87 and a p53-mutated T98G cell, for the same stresses. We ascertained that p53 intensified apoptosis in response to severe DNA damage, not DNA replication stress in glioblastoma. We further asked if p53-mediated apoptosis can be induced by cellular stress other than severe DNA damage. We analyzed two compounds, bortezomib and vorinostat, respective inhibitors of 26S proteasome and histone deacetylase, to evaluate their capacity to activate p53-mediated apoptosis. The cellular stress incited by bortezomib, not vorinostat, activated p53-mediated apoptosis. Next, we asked if the cellular stress generated by combining the two compounds had a synergistic effect on apoptosis. Our results demonstrated that doxorubicin with bortezomib or CFS-1686, or bortezomib with vorinostat have a significant synergistic effect on apoptosis only in p53-wild type cell. Under high stress, p53 translocates from cytosol into the nucleus to cause apoptosis possibly. Together, p53 maximizes apoptosis for cellular stress caused by severe DNA damage, disruption of protein turnover, and for the stress induced by drug combination including doxorubicin with bortezomib or CFS-1686, and bortezomib with vorinostat.

We previously demonstrated that Bim is the main BH3-only protein replacing Bak/Bax from Bcl-xl to activate apoptosis in a p53-independent manner in response to doxorubicin in prostate cancer. However, the comparison of doxorubicin treatment between LNCaP cells carrying p53-wild type and PC3 cells carrying p53-null suggested that p53 might be essential for maximizing apoptosis. Inhibition of ATM did not affect doxorubicin-induced apoptosis. Overexpression of p53 did not affect ABT-263-induced apoptosis and nevertheless, the combination of doxorubicin with ABT-263 induced higher apoptotic responses than did doxorubicin or ABT-263 alone. These results advocated that doxorubicin-induced DNA damage controls p53 function for intensifying apoptosis. Indeed, overexpression of p53 only enhanced apoptosis under conditions of severe DNA damage induced by high concentrations of doxorubicin in LNCaP cells. Immunofluorescence staining showed vague γH2AX foci and enlarged nuclei in LNCaP cells in response to high concentrations of doxorubicin, en route to apoptosis. In addition, our results revealed that the apoptosis in response to DNA replication stress induced by CFS-1686, a catalytic inhibitor of topoisomerase, is p53-independent. Interestingly, the combination of doxorubicin with CFS-1686 generated DNA damage and replication stress simultaneously, resulting in a synergistic apoptotic effect in prostate cancer cells. Thus, we concluded that p53 is a sensor for enhanced apoptosis in response to DNA damage stress, not DNA replication stress, at least in prostate cancer.

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.

Osteoblasts and adipocytes share a common progenitor in bone marrow. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipogenesis. Using a mouse pluripotent mesenchymal cell, D1, as a model, several reports have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. We first examined whether adipogenesis induction in D1 cells is initiated by activation of PPAR-gamma. The results revealed that pioglitazone induces adipogenesis in D1 cells in a dose-dependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a PPAR-gamma antagonist. A PPAR-gamma-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a PPAR-gamma and glucocorticoid receptor pathway. Finally, we analyzed the gene expression profile of D1 by cDNA microarray after treatment with dexamethasone. We found that the expression of several adipogenesis-related genes is highly provoked by this agent.

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.

A yeast genetic screening was developed to isolate androgen receptor (AR) mutants with divergent transactivation characteristics in response to hydroxyflutamide (HF), an active metabolite of flutamide used for prostate cancer treatment. Two mutants carrying the substitution C685Y or E708K were isolated and characterized. Substitution of C685Y for wild-type AR (wtAR) rendered the receptor supersensitive to androgenic activity from HF and female hormones such as 17beta-estradiol (E2) and progesterone (P). Similar effects were observed in the AR mutant, named T876AAR, isolated from LNCaP cells. Surprisingly, we found that C685YAR7, but not T876AAR7, could be activated by casodex (bicalutamide), a nonsteroidal pure antiandrogen, with an induction fold 3- to 5-fold times higher than that for wild type or T876AAR. By contrast, although replacement of E708K for wtAR showed little effect on dihydrotestosterone-mediated transactivation, E708KAR lost its transcriptional response from many other ligands. The effects of ligands on E708KAR could be controlled at the DNA-binding level owing to the finding of a significant decrease in the DNA-binding ability once E708KAR was bound to HF, E2, or P. Together, these results suggest that C685YAR can be a novel tool for assaying the androgenic activity from antiandrogens, and the mechanism revealed from E708KAR could provide a possible explanation for the partial androgen insensitivity syndrome in men with a natural E708KAR mutation.

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

The retinoblastoma protein may function as a tumor suppressor by controlling the progression of the normal cell cycle. Inactivation of Rb has been regarded as an important event in prostate carcinogenesis. However, the detailed mechanism of how Rb is linked to androgen-androgen receptor (A-AR), the major factor in promotion of prostate tumor growth, remains unclear. Using GST-Rb pull down assay and mammalian two-hybrid system, we report here that Rb can bind specifically to AR in an androgen-independent manner. Transient transfection assay demonstrates that cotransfection of AR and Rb can further induce AR transcriptional activity 4-fold in the presence of 1 nM dihydrotestosterone in DU145 cells. Interestingly, cotransfection of Rb and ARA70, the first identified AR coactivator, with AR can additively induce AR transcriptional activity 13-fold (from 5-fold to 64-fold). In conclusion, our discovery that Rb can function as a coactivator to induce AR transcriptional activity in prostate cells may represent the first data to link a negative growth regulatory protein function in a positive manner, by inducing the transcriptional activity of AR.

Androgen alters neurite outgrowth, synaptic organization, and cell survival in various portions of the brain and spinal cord. However, examination of the specific effects of androgen on neurons in vivo has been difficult. Previously, an in vitro model for the effects of estrogen on neurons was developed and characterized, using an estrogen receptor (ER)-transfected PC12 rat pheochromocytoma cell line. This model demonstrated estrogenic regulation of neurite outgrowth, spine formation, and gap junction formation. Similarly, an in vitro model for the effects of androgen on neurons is now described. Wild-type cells (PC12-WT) were stably transfected with an expression vector coding for the full-length cDNA for the human androgen receptor (AR). Resultant clones were isolated, screened for incorporation of vector and expression of AR mRNA and protein, and analyzed for morphologic responses to androgen. PC12-WT, NE09 (ER-negative, AR-negative), SER8 (ER-positive, AR-negative), and AR8 (ER-negative, AR-positive) cells were exposed to 10 ng/ml nerve growth factor (NGF), along with 0-10-7M dihydrotestosterone (DHT) for 2 days. AR8 cells demonstrated an androgen dose-dependent increase in mean neurite length, branch order, and neurite field area, whereas neurite branch segment length and soma area were not affected by androgen. PC12-WT, NE09, and SER8 cells exhibited no alterations in cell morphology with DHT exposure. Because of the synergistic effects of DHT and NGF, the regulation of NGF receptor mRNA by DHT was evaluated; however, no significant induction of either trkA or p75 mRNA expression by androgen was documented. The results suggest that in AR-positive PC12 cells, androgen acts additively with NGF to increase neurite outgrowth; but androgen effects are mediated specifically through branching and arborization. These responses are similar to developmental studies of androgen effects in vivo. Thus, androgen appears to induce an inherent neural morphologic program in AR-containing cells, which increases the receptive field of these cells, increasing the likelihood for interneural communication, although not promoting communication itself. These cell lines will provide a unique in vitro system for studying mechanisms of androgen-neuron interactions.