Gold nanoparticles
(Au NPs) have been thoroughly investigated for
anti-cancer therapy. However, their undesired high gold content remains
a problem when injected into the body for drug delivery applications.
In this report, we made an effort to conjugate the curcumin molecules
on the surface of gold quantum clusters (Au QCs) by a novel in situ
synthesis method which provides an alternative route to not only reduce
the metallic content but also increase the water solubility of curcumin
and the loading efficiency. Here, curcumin itself acts as a reducing
and capping agent for the synthesis of Au QCs. The UV–vis absorption,
fluorescence, transmission electron microscopy, and electrospray ionization
mass spectrometry results confirmed the synthesis of fluorescent Au
QCs. Curcumin-conjugated Au NPs (C-Au NPs) and glutathione (GSH)-conjugated
Au QCs (GSH-Au QCs) were also synthesized to visualize the effect
of particle size and the capping agent, respectively, on the cytotoxicity
to normal and cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay showed that the curcumin-conjugated Au QCs (C-Au QCs)
were less cytotoxic to normal cells while almost the same cytotoxic
to cancer cells in comparison to curcumin itself, which indicates
that curcumin preserves its anticancer property even after binding
to the Au QCs. However, C-Au NPs and GSH-Au QCs did not show any cytotoxicity
against the normal and cancer cells at the concentration used. The
western blot assay indicated that C-Au QCs promote apoptosis in cancer
cells. Further, the in vivo study on severe combined immunodeficiency
mice showed that C-Au QCs also inhibited the tumor growth efficiently
without showing significant toxicity to internal organs.
Background
Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1.
Methods
Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed.
Results
The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner.
Conclusions
SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.
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