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The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in the context of the p53 pathway and the proliferation phenotype in a series of 73 head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also correlated positively with several proteins associated with proliferation, such as cyclin-dependent kinase 2 (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, survivin, and Ki67. The level of VRK1 protein behaves like a proliferation marker in this series of head and neck squamous cell carcinomas. To identify a possible regulatory role for VRK1 and because it regulates gene transcription, the promoters of two genes were studied, CDK2 and SURVIVIN, whose proteins correlated positively with VRK1. VRK1 increases the activity of both the CDK2 and SURVIVIN gene promoters. The expression of VRK1 was analyzed in the context of regulators of the G1-S transition. VRK1 protein levels increase in response to E2F1 and are reduced by retinoblastoma and p16. These data suggest that VRK1 might play a role in cell cycle regulation and is likely to represent the beginning of a new control mechanism of cell cycle, particularly late in the G1-S phase.

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.

Background: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2–8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. Methods: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. Results: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.05 and 4.5 nmol × h/mL for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 90:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 19 and 28 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.5:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. Conclusion: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX. [ABSTRACT FROM AUTHOR]

It is known that tRNAs play an essential role in genetic information transfer from DNA to protein. The maturation of tRNA precursors is performed by the endoribonuclease RNase P, which classically consists of a main RNA segment and accessory proteins. However, the newly identified human mitochondrial RNase P-like protein (MRPP123) complex is unique in that it is composed of three proteins without RNA. Here, we determined the crystal structure of MRPP123 complex subunit 3 (MRPP3), which is thought to carry out the catalytic reaction. A detailed structural analysis in combination with biochemical assays suggests that MRPP3 is in an auto-inhibitory conformation in which metal ions that are essential for catalysis are excluded from the active site. Our results indicate that further regulation is necessary to rearrange the conformation of the active site of MRPP3 and trigger it, thus providing important information to understand the activation of MRPP123. [ABSTRACT FROM AUTHOR]

Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G2/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5-15-fold) within 4 h without G2/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G2/M cells were significantly increased at later times. Interestingly, 3 nM taxol induces survivin as effectively as 300 nM and more effectively than 3000 nM. As a result, 3 nM taxol is ineffective at inducing cell death. However, inhibition of taxolmediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1480 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxolmediated induction of survivin is an early event and independent of taxol-mediated G2/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics. [ABSTRACT FROM AUTHOR]