Chanlu Xie

p27(Kip1) signaling: Transcriptional and post-translational regulation.

p27(Kip1) is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27(Kip1) allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27(Kip1) protein levels, in particular nuclear expressed p27(Kip1), is implicated in both disease progression and poor prognosis in a variety of cancers. p27(Kip1) expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27(Kip1) protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27(Kip1) at Thr(187) and Ser(10) is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser(10) phosphorylated p27(Kip1) is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27(Kip1) in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27(Kip1) localization and/or block its degradation.

The effects of 56MESS on mitochondrial and cytoskeletal proteins and the cell cycle in MDCK cells

BACKGROUND 56MESS has been shown to be cytotoxic but the mode of this action is unclear. In order to probe the mechanism of action for 56MESS, MDCK cells were utilised to investigate the effect on treated cells. RESULTS IC50 values for 56MESS and cisplatin in the MDCK cell line, determined by a SRB assay, were 0.25 ± 0.03 and 18 ± 1.2 μM respectively. In a preliminary study, cells treated with 56MESS displayed no caspase-3/7 activity, suggesting that the mechanism of action is caspase independent. Protein expression studies revealed an increase the expression in the MTC02 protein associated with mitochondria in cells treated with 56MESS and cisplatin. Non-synchronised 56MESS-treated cells caused an arrest in the G2/M phase of the cell cycle, in comparison to the S phase arrest of cisplatin. In G0/G1 synchronised cells, both 56MESS and cisplatin both appeared to arrest within the S phase. CONCLUSIONS these results suggest that 56MESS is capable of causing cell-cycle arrest, and that mitochondrial and cell cycle proteins may be involved in the mode of action of cytotoxicity of 56MESS.

Ubiquitin-conjugating enzyme E2C: A potential cancer biomarker

The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy.

Prostate-Regenerating Capacity of Cultured Human Adult Prostate Epithelial Cells

Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience, we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study, benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma, which were then cultured in the medium designed for prostate epithelial and stromal cells, respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells, the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus, the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium.

Proliferating cell unclear antigen-associated factor (PAF15): A novel oncogene

Proliferating cell nuclear antigen (PCNA)-Associated Factor (PAF15) is a small protein containing a PCNA interacting motif and sequences for association with ubiquitin enzymes. In interaction with PCNA, PAF15 plays a key role in recruiting DNA replicative polymerase by double monoubiquitination at Lys(15) and Lys(24). Under DNA damage conditions, PAF15 regulates the switch from DNA replicative polymerase to translesion synthesis polymerase in order to bypass the replication-blocking lesions. Overexpression of PAF15 promotes the repair of ultraviolet-induced DNA damage and prevents cell death, whereas attenuation of PAF15 decreases DNA replication and cell survival. Ectopic expression of PAF15 in mouse fibroblasts increases colony formation and tumourigenicity. PAF15 is aberrantly increased in various human malignancies with poor prognosis. Collectively, PAF15 may contribute to carcinogenesis and represents one of the potential therapeutic targets in the treatment of cancer.

Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the 5-year survival rate of advanced HCC is < 10%. Guttiferone K (GUTK) isolated from the Garcinia genus inhibited HCC cells migration and invasion in vitro and metastasis in vivo without apparent toxicity. Proteomic analysis revealed that actin-binding protein profilin 1 (PFN1) was markedly increased in the presence of GUTK. Over-expression of PFN1 mimicked the effect of GUTK on HCC cell motility and metastasis. The effect of GUTK on cell motility was diminished when PFN1 was over-expressed or silenced. Over-expression of PFN1 or incubation with GUTK decreased F-actin levels and the expression of proteins involved in actin nucleation, branching and polymerization. Moreover, a reduction of PFN1 protein levels was common in advanced human HCC and associated with poor survival rate. In conclusion, GUTK effectively suppresses the motility and metastasis of HCC cells mainly by restoration of aberrantly reduced PFN1 protein expression.

Guttiferone K impedes cell cycle re-entry of quiescent prostate cancer cells via stabilization of FBXW7 and subsequent c-MYC degradation.

Cell cycle re-entry by quiescent cancer cells is an important mechanism for cancer progression. While high levels of c-MYC expression are sufficient for cell cycle re-entry, the modality to block c-MYC expression, and subsequent cell cycle re-entry, is limited. Using reversible quiescence rendered by serum withdrawal or contact inhibition in PTENnull/p53WT (LNCaP) or PTENnull/p53mut (PC-3) prostate cancer cells, we have identified a compound that is able to impede cell cycle re-entry through c-MYC. Guttiferone K (GUTK) blocked resumption of DNA synthesis and preserved the cell cycle phase characteristics of quiescent cells after release from the quiescence. In vehicle-treated cells, there was a rapid increase in c-MYC protein levels upon release from the quiescence. However, this increase was inhibited in the presence of GUTK with an associated acceleration in c-MYC protein degradation. The inhibitory effect of GUTK on cell cycle re-entry was significantly reduced in cells overexpressing c-MYC. The protein level of FBXW7, a subunit of E3 ubiquitin ligase responsible for degradation of c-MYC, was reduced upon the release from the quiescence. In contrast, GUTK stabilized FBXW7 protein levels during release from the quiescence. The critical role of FBXW7 was confirmed using siRNA knockdown, which impaired the inhibitory effect of GUTK on c-MYC protein levels and cell cycle re-entry. Administration of GUTK, either in vitro prior to transplantation or in vivo, suppressed the growth of quiescent prostate cancer cell xenografts. Furthermore, elevation of FBXW7 protein levels and reduction of c-MYC protein levels were found in the xenografts of GUTK-treated compared with vehicle-treated mice. Hence, we have identified a compound that is capable of impeding cell cycle re-entry by quiescent PTENnull/p53WT and PTENnull/p53mut prostate cancer cells likely by promoting c-MYC protein degradation through stabilization of FBXW7. Its usage as a clinical modality to prevent prostate cancer progression should be further evaluated.

Food Extracts Consumed in Mediterranean Countries and East Asia Reduce Protein Concentrations of Androgen Receptor, Phospho-Protein Kinase B, and Phospho-Cytosolic Phospholipase A2α in Human Prostate Cancer Cells

Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells

Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.