[Kamat2010]

Ubiquitin is an essential protein present abundantly within the body and is used for the normal functioning and degradation of other proteins within the cell by covalent modification. The ubiquitin acts along with the Proteasome complex, which is multicatalytic proteinase. The Ubiquitin-Proteasome complex plays a very important role in the turnover of the different cellular proteins, which are usually degraded or damaged in nature, by the Ubiquitin-Protease pathway (UPP). The UPP has no effect in general on other normal proteins and the proteins, which are to be degraded, are targeted by the protease system by conjugation with ubiquitin.

The Ubiquitin-Protease system is responsible for the regulation of the mitosis and other important steps in the regulation of the cell cycle by the degradation of the damaged and unnecessary proteins, thereby preventing the accumulation of the unnecessary proteins within the cell. Thus, ubiquitin-protease system plays an important role in the maintenance of cellular homeostasis within the body by regulating the different signalling pathways, antigen-rocognition, etc. The accumulated damaged proteins within the cell cause apoptosis i.e. programmed cell death. This apoptosis is prevented by the Ubiquitin-protease system. Hence, the study of this system was undertaken to see if the inhibition of this system has any effect in promoting apoptosis of tumor cells thereby helping in cancer therapy. This could target either inhibition of the ubiquitination of the proteins to be degraded or the inhibition of the proteasomes, both of which cause protein degradation inhibition resulting ultimately in apoptosis.

The large sub-cellular organelle, which is multi-subunit protein complex, is the site for the ATP-dependent Ubiquitin mediated protein degradation within the cell and is known as Proteasome. The Proteasome is also known as 26S proteasome and consists of two subunits: 20S subunit, which is the catalytic subunit and the 19S subunit, which is the regulatory subunit. The 19S subunit consists of ATPases, which hydrolyse ATP thereby causing change in the conformation of the 20S subunit that binds to the unfolded substrate. The isopeptidase in the 19S subunit causes the cleavage of the ubiquitin molecules, which are then available for the ubiquitination of the subsequent proteins ready for degradation. Many research studies have proved that many inhibitors are available for the inhibition of the proteasome complexes, which ultimately lead to apoptosis. Some studies have proved the effectiveness of proteasome inhibition on the treatment of the multiple myeloma. Unlike other cells, multiple myeloma is susceptible to the accumulation of the unwanted proteins like large number of immunoglobulin chains as they are derived from the antibody producing cells. Due to this accumulation, the inhibition of the proteasome with the use of inhibitors causes the programmed cell death or apoptosis of the multiple myeloma cells. The normal cells are not affected in this case with the use of the proteasome inhibitors as the normal cells do not accumulate unwanted proteins in large quantities due to less need for proteasomal action. Moreover, the normal cells have alternative methods of removal of the damaged proteins like autophagy, whereby the unnecessary, aggregated proteins are transported to the lysosomes for removal, which helps in the removal of these proteins. Thus, it can be seen that this method of autophagy prevents apoptosis in some tumor cells. Hence, the use of inhibitors of autophagy along with the proteasome inhibitors may help in the apoptosis of the tumor cells better.

In some cases like breast cancer therapy, indirect function of the ubiquitin-protease system is seen. It is seen that the use of certain compounds was found to increase the ubiquitin mediated degradation of proteins required for normal cell function thereby causing apoptosis. The use of camptothecins was found to increase the proteolytic degradation of the topoisomerases by the ubiquin-mediated mechanisms. In some other cases, the oestrogen receptors were degraded by the UPP in the postmenopausal patients suffering from breast cancer with the use of certain drugs.

In this way, it can be seen that the UPP plays an important role in the different areas of cancer therapy. Extensive research is going on in this field and the exact mechanism by which the inhibition or the overfunctioning of the protease system plays a role in the programmed cell death of the tumor or cancer cells is yet to be analysed.

[mtwalsh01]

Proteasomal inhibition and myeloma

The original article in this thread addresses the ubiquitin-protease system in cancer therapy. The article describes the proteasome, the cellular structure responsible for ATP-dependent degradation of ubiquitin-tagged damaged proteins. The article also alludes to the effectiveness of proteasome inhibition on the treatment of the multiple myeloma. In fact, a proteasome inhibitor called bortezomib has already been approved for treatment of both multiple myeloma and mantle cell lymphoma. However, the situation is not straightforward, despite the acceptance of bortezomib as an important weapon in the armoury of myeloma therapy. Matters are complicated by various problems associated with bortezomib, including emerging drug resistance issues, some normal cell toxicity and inconvenience to patients due to administration by intravenous injection. Thus, research continues on other potential proteasome inhibitors that may be safer and more conveniently taken.

A review from the University of Michigan, for example, has summarised current research on enzymes upstream of the proteasome, namely the Cullin-RING Ligases (CRLs) as attractive targets for multiple myeloma therapy. These are the largest family of E3 ubiquitin ligases, the enzymes that tag damaged proteins with ubiquitin to identify them for proteasomal degradation. Activity of CRLs is dynamically regulated, requiring both the RING component and cullin neddylation. A small molecule indirect inhibitor of CRL named MLN4924, which blocks cullin neddylation, is currently in phase I clinical trials and other inhibitors are under investigation.

Other studies are focusing more on alternative proteasome inhibitors. A group from The Netherlands Cancer Institute in Amsterdam investigated the possibilities of the novel proteasome inhibitor delanzomib and compared its activity to bortezomib both in vitro and in a preclinical multiple myeloma model. Using fluorogenic substrates and a recently developed fluorescent proteasome activity probe as indicators of inhibition of proteasome activity, the group found that both inhibitors inhibited proteasome activity to a similar extent in cell lines in vitro. In the pre-clinical model, while both inhibitors exhibited similar inhibition of the proteasome in normal tissues, delanzomib was twice as potent as bortezomib in terms of tumour proteasome activity. Further clinical testing of delanzomib was indicated.

Meanwhile a study from Jichi Medical University was concerned with the inconvenience of the necessity of administering Bortezomib by intravenous injection as well as drug resistance. This study focused on the necessity of developing orally active proteasome inhibitors. K-7174 is a member of the homopiperazine derivatives, a class of proteasome inhibitors with a different mode of action than bortezomib. K-7174 acts by repressing transcription of class I histone deacetylases (HDAC1, -2, and -3) via caspase-8-dependent degradation of Sp1. In a mouse myeloma model, K-7174 had a therapeutic effect, which was stronger when administered orally than intravenously. No obvious cytotoxic side-effects were observed. In terms of drug resistance to bortezomib, K-7174 was also able to kill bortezomib-resistant myeloma cells carrying a β5-subunit mutation in vivo and primary cells from a patient resistant to bortezomib. HDAC inhibitors enhanced K-7174 activity with an increase in histone acetylation. Thus K-7174 has potential as a proteasome inhibitor that can be taken orally and which may overcome drug resistance issues associated with bortezomib resistance. Clinical testing may yield promising results.

Sources

BERKERS, C.R. et al., 2012. Probing the specificity and activity profiles of the proteasome inhibitors bortezomib and delanzomib. Molecular Pharmaceutics, 9(5), pp. 1126-1135

KIKUCHI, J. et al., 2013. The Novel Orally Active Proteasome Inhibitor K-7174 Exerts Anti-myeloma Activity in Vitro and in Vivo by Down-regulating the Expression of Class I Histone Deacetylases. The Journal Of Biological Chemistry, 288(35), pp. 25593-25602

ZHAO, Y. and SUN, Y., 2013. Cullin-RING Ligases as attractive anti-cancer targets. Current pharmaceutical design, 19(18), pp. 3215-3225