Category ME L18 Proteasome Inhibition and Apoptosis by DSF-analogue #39 in MDA-

MB-231 cells

Abstract Copper is one of the most crucial elements in tumor cell growth because it

is required for several molecular processes in angiogenesis, which is

essential for tumors to spread and metastasize. It is known that there are

higher concentrations of copper in cancer cells than normal cells creating

a differentiation between these cells, which provides a mechanism of

using copper to target only cancer cells as an anticancer strategy. In

recent studies, it has been found that Disulfiram (DSF), a clinically used

medicine for the treatment of alcoholism, is able to bind to copper forming a

potent DSF- Cu conjugate that inhibits proteasome activity and induces

apoptosis in human breast cancer cells. However, clinical use of DSF for

the treatment of cancer is limited because of its toxicity to normal cells. As

well, DSF irreversibly inhibits aldehyde dehydrogenase and other

enzymes, causing toxicities, which makes it a disadvantage considering its

anticancer effects. In this study, I am trying to investigate whether a

variety of DSF analogues are able to bind to copper and form a complex

that produces apoptotic cancer cell death through the inhibition of the

proteasome, as well as have less toxicity to normal cells.



MDA-MB-231 breast cancer cells were cultured, and incubated with

different concentrations of the DSF analogues coupled with copper. MTT

assays were taken to determine the effects of the compounds and copper

to the proliferation of the breast cancer cells. A Western Blot Analysis

was conducted to determine proteasome inhibition, followed by a

Proteasomal Chymotrypsin- like activity assay demonstrating the effects of

the DSF analogues-copper mixtures on the proteasome. A cellular

morphology analysis was performed as well.



It has been demonstrated that designated DSF analogue #39 with copper

was the most potent compound-copper conjugate with ~100% inhibition of

MDA-MB-231 cells at concentrations of 1 um – 10 um, even stronger than

DSF-Cu, which was used as a positive control. For breast cancer cells

treated with #39-Cu, there was inhibition of the proteasomal CT-like

activity, and through Western blot an accumulation of ubiquitinated proteins

and PARP cleavage was seen. Preliminary tests also showed that the

DSF analogue #39 has similar toxicity to the MCF-10A normal breast cell

line as the DSF-Cu complex.



In Conclusion, the designated DSF analogue #39 with copper had potent

anti-proliferation effects through the inhibition of proteasome activity in

cultured breast cancer cells even greater than the DSF-Cu complex, while

it had similar toxicity to normal cells. However, since DSF has multiple

toxicities, further toxicity studies of #39-Cu should be conducted using a

different model. Overall, the data presented here suggests that the DSF-

analogue #39 after coupled with Cu, is a promising complex for further

tests to be a useful anti-tumor drug.




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