Delivery of PTEN via a novel gene microcapsule sensitizes prostate cancer cells to irradiation

The tumor suppressor gene MMAC/PTEN located on chromosome10q23.3 has dual phosphatase activity in the phosphoinositide-3-kinase signaling pathway and inhibits Akt activation, a serine-threonine kinase, which is involved in proliferative and antiapoptotic pathways. Furthermore, MMAC/PTEN is frequently inactivated in a variety of tumors including prostate cancer. In this study, we generated a new type of gene transfer drug, GelaTen, which is a microsphere of cationized gelatin hydrogels incorporating PTEN plasmid DNA. Using our previously reported radiation-resistant PC3-Bcl-2 human prostate cancer cells (PTEN deleted), we examined the efficacy of GelaTen to force the expression of PTEN in vivo to inhibit tumor growth after intratumoral injection alone or with irradiation. Combinational therapy with GelaTen and irradiation improved both the in vitro and in vivo efficacy of growth inhibition compared with GelaTen or irradiation alone. These data show that GelaTen gene therapy, enabling radiosensitization, can potentially treat prostate cancers that have MMAC/PTEN gene alterations associated with radioresistance.     

Transient reduction of PTI-1 expression by short interfering RNAs inhibits the growth of human prostate cancer cell lines

The prostate tumor-inducing gene 1 (PTI-1) was originally identified by differential ribonucleic acid (RNA) display in human prostate carcinoma. PTI-1 is expressed in human prostate carcinoma but not in benign prostate hypertrophy or normal prostate tissue. PTI-1 may be a member of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate. To investigate the role of PTI-1 in human prostate carcinoma, we constructed three different short interfering RNA (siRNA) vectors (pSilencer3.1-neo-Yu Lei [YL]1-2, -YL3-4 and -YL5-6), each of which was transfected into DU145 and PC3 human prostate cancer cell lines. Among these siRNAs, only pSilencer3.1-neo-YL1-2 could almost completely block the expression of PTI-1 in these two cell lines. The growth of the cell lines was then evaluated after transfection. The proliferation rate was retarded in DU145 and PC3 cells transfected with pSilencer3.1-neo-YL1-2, compared with the cells transfected with a control vector; namely, about 88.6% of DU145 and 80.2% of PC3 cancer cells were blocked at the G1 phase when transfected with pSilencer3.1-neo-YL1-2, compared to 62.0% in DU145 cells and 51.7% in PC3 cells, transfected with the control vector. Moreover, 68.3% of DU145 cells and 72.3% of PC3 cells were induced into apoptosis, while in control transfection, the population was 26.6% in DU145 cells and 28.4% in PC3 cells. These results indicate that blocking PTI-1 expression can inhibit the growth of certain prostate cancer cell lines. We suggest that PTI-1 may serve as a target for the gene-based therapy of human prostate carcinoma.     

Modulation of Akt/mTOR signaling overcomes sunitinib resistance in renal and prostate cancer cells

Tyrosine kinase inhibitors exhibit impressive activity against advanced renal cell carcinoma. However, recent clinical studies have shown an equivocal response to sunitinib in patients with castration-resistant prostate cancer. The tumor suppressor PTEN acts as a gatekeeper of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR cell-survival pathway. Our experiments showed that PTEN expression inversely correlates with sunitinib resistance in renal and prostate cancer cells. Restoration of PTEN expression markedly increases sensitivity of tumor cells to sunitinib both in vitro and in vivo. In addition, pharmacologic manipulation of PI3K/Akt/mTOR signaling with PI3K/mTOR inhibitor, GDC-0980, mTOR inhibitor, temsirolimus, or pan-Akt inhibitor, GSK690693, was able to overcome sunitinib resistance in cancer cells. Our findings underscore the importance of PTEN expression in relation to sunitinib resistance and imply a direct cytotoxic effect by sunitinib on tumor cells in addition to its antiangiogenic actions.     

低频低功率超声联合微泡对DU145细胞及PC3细胞早期凋亡的影响

目的比较低频低功率超声联合微泡造影剂对人前列腺癌DU145细胞与PC3细胞早期凋亡率的不同影响。方法使用低频低功率超声连续波辐照人前列腺癌DU145细胞和PC3细胞悬液60 s,实验中两种细胞系各分为4组:空白对照组(A组)、单纯微泡组(B组)、单纯超声组(C组)和超声联合微泡组(D组),辐照后细胞继续培养24 h,流式细胞仪检测细胞早期凋亡情况,透射电镜观察细胞形态改变。结果两种细胞D组的早期细胞凋亡率明显高于其余各组(P﹤0.01),C组亦均高于A组(P﹤0.05),A、B组之间差异无统计学意义;两种细胞经相同处理后,C、D组PC3细胞的早期细胞凋亡率均高于DU145细胞(P﹤0.01)。透射电镜下两种细胞D组可见癌细胞体积变小变圆,空泡增多,有明显的凋亡小体形成,PC3细胞内还可见大量的自噬体出现。结论低频低功率超声联合微泡造影剂能明显诱导人前列腺癌细胞的早期凋亡,且对PC3细胞早期细胞凋亡作用强于DU145细胞。     

低频超声联合微泡增强脂质体介导Racl—shRNA质粒转染前列腺癌细胞及对其生物学行为的影响

目的研究低频超声联合微泡增强脂质体介导的Racl-shRNA质粒转染前列腺癌PC3、DUl45细胞,并探索其对前列腺癌细胞凋亡、侵袭能力的影响。方法基础情况下Westernblot检测RWPE-1细胞、PC3细胞和DUl45细胞的Rac1蛋白表达。根据不同处理方式将细胞分为四组：PC3细胞对照组（A组）、超声加微泡联合脂质体转染PC3细胞组（B组）、DUl45细胞对照组（c组）及超声加微泡联合脂质体转染DUl45细胞组（D组）。通过蛋白质体外结合实验技术检测各组Racl蛋白的活性及表达;流式细胞仪检测各组细胞早期凋亡情况;细胞侵袭实验检测各组细胞侵袭能力。结果基础情况下前列腺癌PC3、DUl45细胞Racl蛋白表达均较正常前列腺细胞RWPE-1增加,差异均有统计学意义（均P〈0．01）;低频超声携微泡介导Rac-1shRNA质粒转染细胞后,B组Racl蛋白表达量较A组表达减少,D组表达量较C组减少,差异均有统计学意义（均P〈0．01）;早期细胞凋亡检测显示,B组高于A组,D组高于C组,差异均有统计学意义（均P〈0．01）;同期细胞侵袭力实验显示,B组穿膜细胞数少于A组,D组的穿膜细胞数少于C组,差异均有统计学意义（均P〈0．01）。结论低频超声联合微泡可促进脂质体介导的Racl-shRNA质粒转染人雄激素非依赖型前列腺癌细胞;通过抑制PC3、DUl45细胞中Racl蛋白表达可以促进癌细胞的早期细胞凋亡,同时可抑制癌细胞的侵袭能力。     

z-Guggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo.

Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer.     

Combined targeting of epidermal growth factor receptor and hedgehog signaling by gefitinib and cyclopamine cooperatively improves the cytotoxic effects of docetaxel on metastatic prostate cancer cells

The epidermal growth factor receptor (EGFR) and hedgehog cascades provide a critical role in prostate cancer progression and contribute to the resistance to clinical therapies and disease relapse. Therefore, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective inhibitors of EGFR tyrosine kinase and smoothened hedgehog signaling element, gefitinib and cyclopamine, with a current chemotherapeutic drug used in the clinics, docetaxel, on some metastatic prostate cancer cell lines. Immunohistochemical analyses revealed that sonic hedgehog (SHH) expression was enhanced in 39% of primary prostatic adenocarcinomas (Gleason scores 4-10) compared with the corresponding normal tissues of the same prostate gland from 32 prostate cancer patients. The confocal microscopy and Western blot analyses have also indicated the high expression levels of SHH and EGFR in metastatic LNCaP, DU145, and PC3 cells. Moreover, the results revealed that the drugs, alone or in combination, at lower concentrations inhibited the growth of EGF plus SHH-stimulated and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145, and PC3 cells. Importantly, the combined docetaxel, gefitinib, and cyclopamine also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. The cytotoxic effects induced by these drugs in PC3 cells seem to be mediated in part through the cellular ceramide production and activation of caspase cascades via a mitochondrial pathway and the release of cytochrome c into the cytosol. Additionally, the combined agents were more effective at suppressing the invasiveness of PC3 cells through Matrigel in vitro than the single drugs. These findings indicate that the combined use of inhibitors of EGF-EGFR and hedgehog signaling with docetaxel could represent a more promising strategy for treatment in patients with metastatic and androgen-independent prostate cancer.     

Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells

The up-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is prevalent in many cancers. This phenomenon makes PI3K and Akt fruitful targets for cancer therapy and/or prevention because they are mediators of cell survival signaling. Although the suppression of phospho-Akt by selenium has been reported previously, little information is available on whether selenium modulates primarily the PI3K-phosphoinositide-dependent kinase 1 (PDK1) side of Akt phosphorylation or the phosphatase side of Akt dephosphorylation. The present study was aimed at addressing these questions in PC-3 prostate cancer cells which are phosphatase and tensin homologue-null. Our results showed that selenium decreased Akt phosphorylation at Thr308 (by PDK1) and Ser473 (by an unidentified kinase); the Thr308 site was more sensitive to selenium inhibition than the Ser473 site. The protein levels of PI3K and phospho-PDK1 were not affected by selenium. However, the activity of PI3K was reduced by 30% in selenium-treated cells, thus discouraging the recruitment of PDK1 and Akt to the membrane due to low phosphatidylinositol-3,4,5-trisphosphate formation by PI3K. Consistent with the above interpretation, the membrane localization of PDK1 and Akt was significantly diminished as shown by Western blotting. In the presence of a calcium chelator or a specific inhibitor of calcineurin (a calcium-dependent phosphatase), the suppressive effect of selenium on phospho-Akt(Ser473) was greatly reduced. The finding suggests that selenium-mediated dephosphorylation of Akt via calcineurin is likely to be an additional mechanism in regulating the status of phospho-Akt.     

A novel histone deacetylase (HDAC) inhibitor MHY219 induces apoptosis via up-regulation of androgen receptor expression in human prostate cancer cells

Histone deacetylase (HDAC) inhibitors are a new class of anticancer agents that act by inhibiting cancer cell proliferation and inducing apoptosis in various cancer cell lines. To investigate the anticancer effect of a novel histone deacetylase (HDAC) inhibitor MHY219, its efficacy was compared to that of suberoylanilide hydroxamic acid (SAHA) in human prostate cancer cells. The anticancer effects of MHY219 on cell viability, HDAC enzyme activity, cell cycle regulation, apoptosis and other biological assays were performed. MHY219 was shown to enhance the cytotoxicity on DU145 cells (IC₅₀, 0.36 μM) when compared with LNCaP (IC₅₀, 0.97 μM) and PC3 cells (IC₅₀, 5.12 μM). MHY219 showed a potent inhibition of total HDAC activity when compared with SAHA. MHY219 increased histone H3 hyperacetylation and reduced the expression of class I HDACs (1, 2 and 3) in prostate cancer cells. MHY219 effectively increased the sub-G1 fraction of cells through p21 and p27 dependent pathways in DU145 cells. MHY219 significantly induced a G2/M phase arrest in DU145 and PC3 cells and arrested the cell cycle at G0/G1 phase in LNCaP cells. Furthermore, MHY219 effectively increased apoptosis in DU145 and LNCaP cells, but not PC3 cells, according to Annexin V/PI staining and Western blot analysis. These results indicate that MHY219 is a potent HDAC inhibitor that targets regulating multiple aspects of cancer cell death and might have preclinical value in human prostate cancer chemotherapy, warranting further investigation.     

Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells

Berberine, a naturally occurring isoquinoline alkaloid, has been shown to possess anti-inflammatory and antitumor properties in some in vitro systems. Here, we report that in vitro treatment of androgen-insensitive (DU145 and PC-3) and androgen-sensitive (LNCaP) prostate cancer cells with berberine inhibited cell proliferation and induced cell death in a dose-dependent (10-100 micromol/L) and time-dependent (24-72 hours) manner. Treatment of nonneoplastic human prostate epithelial cells (PWR-1E) with berberine under identical conditions did not significantly affect their viability. The berberine-induced inhibition of proliferation of DU145, PC-3, and LNCaP cells was associated with G1-phase arrest, which in DU145 cells was associated with inhibition of expression of cyclins D1, D2, and E and cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 proteins, increased expression of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27), and enhanced binding of Cdk inhibitors to Cdk. Berberine also significantly (P < 0.05-0.001) enhanced apoptosis of DU145 and LNCaP cells with induction of a higher ratio of Bax/Bcl-2 proteins, disruption of mitochondrial membrane potential, and activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase. Pretreatment with the pan-caspase inhibitor z-VAD-fmk partially, but significantly, blocked the berberine-induced apoptosis, as also confirmed by the comet assay analysis of DNA fragmentation, suggesting that berberine-induced apoptosis of human prostate cancer cells is mediated primarily through the caspase-dependent pathway. The effectiveness of berberine in checking the growth of androgen-insensitive, as well as androgen-sensitive, prostate cancer cells without affecting the growth of normal prostate epithelial cells indicates that it may be a promising candidate for prostate cancer therapy.     

Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo

The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers.     

Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapamycin, was used to study the effects of mTOR inhibition, as the role of mTOR inhibition in enhancing radiation remains unexplored. RAD001 decreased clonogenic cell survival in both breast cancer cell lines MDA-MB-231 and MCF-7, although the effect is greater in MDA-MB-231 cells. Irradiation induced Akt and mTOR signaling, and this signaling is attenuated by RAD001. The radiation-induced signaling activation is mediated by PI3K because inhibition of PI3K with LY294002 inhibited the increase in downstream mTOR signaling. Additionally, caspase-dependent apoptosis is an important mechanism of cell death when RAD001 is combined with 3 Gy radiation, as shown by induction of caspase-3 cleavage. An increase in G(2)-M cell cycle arrest was seen in the combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study. We conclude that RAD001 attenuates radiation-induced prosurvival Akt/mTOR signaling and enhances the cytotoxic effects of radiation in breast cancer cell models, showing promise as a method of radiosensitization of breast cancer.     

Sensitization of breast cancer cells to radiation by trastuzumab

HER2, a member of the human epidermal growth factor (EGF) receptor family, not only plays important roles in the progression of breast cancer tumorigenesis and metastasis, but may protect cancer cells from conventional cytotoxic therapies as well. In the current study, we evaluated the effect of targeting HER2 on radiosensitization of human breast cancer cells. Using six breast cancer cell lines with various levels of HER2 (BT474, SKBR3, MDA453, MCF7, ZR75B, and MDA468), we found that trastuzumab (Herceptin), a humanized monoclonal antibody that may inhibit breast cancer cell proliferation but does not induce apoptosis when used alone, enhanced radiation-induced apoptosis of the cells in a HER2 level-dependent manner. We furthered this study in MCF7 cells transfected for high levels of HER2 (MCF7HER2). Compared with parental or control vector-transfected MCF7 cells, MCF7HER2 cells showed increased phosphorylation of at least two important HER2 downstream molecules, protein kinase B/Akt and mitogen-activated protein kinase (MAPK), and increased resistance to radiotherapy, as shown by reduced induction of apoptosis and increased cell clonogenic survival after radiation. Exposure of the cells to trastuzumab down-regulated the levels of HER2 and reduced phosphorylation levels of Akt and MAPK in MCF7HER2 cells, and sensitized these cells to radiotherapy. When specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and MAPK kinase (MEK) pathways were used, we found that exposure of MCF7HER2 cells to the PI3-K inhibitor LY294002 inhibited Akt phosphorylation and radiosensitized the cells, whereas the radiosensitization effect by the MEK inhibitor PD98059 was relatively weaker, albeit the phosphorylation of MAPK was reduced by PD98059 treatment. Our results indicate that the PI3-K pathway might be the major pathway for trastuzumab-mediated radiosensitization of breast cancer cells.     

Gene therapy approach in prostate cancer cells using an active Wnt signal.

BACKGROUND: Functional activation of beta-catenin/T-cell factor (Tcf) signaling plays an important role in the early events of carcinogenesis. In past recent years accumulated evidence has demonstrated a significant role for the Wnt pathway in the development and progression of human prostate cancer. The objective of the current study was to use a gene-targeting approach to selectively kill human prostate cancer cells with activated beta-catenin/Tcf signaling. METHODS: A recombinant adenovirus that carries a lethal gene (PUMA) under the control of a beta-catenin/T-cell factor (Tcf)-responsive promoter (Ad-TOP-PUMA), was used to selectively target human prostate cancer cells (PC-3) in which the beta-catenin/Tcf pathway is activated, and compared its killing efficiency in cancer cells in which this pathway is inactive (DU145 cells). Ad-FOP-PUMA, carrying a mutant Tcf binding site, was used as a control virus. Cell viability was measured by methylene blue assay, and the level of beta-catenin/Tcf activity was measured by luciferase assay. RESULTS: The Ad-TOP-PUMA adenovirus inhibited PC-3 cell growth in a dose and time-dependent fashion, but did not had any effect on DU145 cell growth. CONCLUSIONS: Selective targeting of prostate cancer cells with the activated beta-catenin pathway may be a novel and effective therapy in prostate cancer.