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. 2016 Jun 21:6:28196.
doi: 10.1038/srep28196.

Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

Santosh K Misra  1 , Mao Ye  1 , Fatemeh Ostadhossein  1 , Dipanjan Pan  1
Affiliations

Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

Santosh K Misra et al. Sci Rep. .

Abstract

Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis.

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Figures

Figure 1
Figure 1. Repairing cancer cell suicide mechanism (programmed cell death).
A modulation of PDK by nano-enabled delivery of haloacetates. (A) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/DMAP, RT, 12 h, (B) polyethylene glycol cetyl ether, self-assembly, H2O:THF (4:1, v/v).
Figure 2
Figure 2. In-silico studies to identify lead structure and key interactions.
(A) Key identified interactions of DCA to the residues of the _target DCA binding pocket of 2BU8; (B) docking pose of dichloroacetate (Cyan) to binding pocket of 2BU8; (C) key interaction of docking pose pose of DCA with the _target; (D) superimposition of docking pose of various haloacetates, with dichloroacetate (Cyan).
Figure 3
Figure 3. Physico-chemical properties of pro-haloacetate nanoparticles.
(A) Number avg. hydrodynamic diameter distribution; (B) Surface zeta potential distribution; Representative TEM images of (C) Pro-DCA-NP; (D,F) Pro-MCA-NP and (F) lipid-NPs (4% uranyl acetate) with comparative anhydrous diameter (G) and Representative EDX elemental distribution histograms from (H) pro-haloacetate nanoparticles (I) representative XRD pattern from lipid-NP and Pro-MCA-NP.
Figure 4
Figure 4. Cell growth inhibition studied by MTT assay.
Assay performed in (A) Panc1; (B) MCF-7; (C) MDA-MB231 and (D) BT549 cells treated with formulations at concentration ranging from 10–500 μM for 48 h and (E) BT549 treated for 72 h. Representative histograms from propidium iodide stained MDA-MB231 and MCF-7 (F-O) for quantifying % apoptotic population (yellow) after treatment with (G,L) Pro-DCA-NP; (H,M) Pro-MCA-NP; (I,N) DCA and (J,O) Lipid-NP formulations at 50 μM for 48 h while (F,K) represent non treated cells. The %apoptotic population compared in (P) MDA-MB231 and (Q) MCF-7 cells. (R) Gel electrophoresis performed on fragmented genomic DNA extracted from MCF-7 (Lane 1–6) and MDA-MB231 (Lane 8–13) cells while lane 7 represent 1 kb ladder. Genomic DNA was extracted from cells treated with (Lane 1, 8) Pro-MCA-NP; (Lane 2, 9) DCA; (Lane 3, 10) untreated cells; (Lane 4, 11) DBA; (Lane 5, 12) MCA and (Lane 6, 13) Pro-DCA-NP formulations at 50 μM in MDA-MB231 and 200 μM in MCF-7 due to different IC50 levels in both the cells for 72 h. (S) Drug release study performed on Pro-MCA-NPs against acetate buffer (pH 4.6); (T) circular dichroism performed for PDK2 protein against added MCA and (U) mitochondrial enrichment of MCA calculated by estimating chloride provide significant input on role of MCA after being released from Pro-MCA-NP.
Figure 5
Figure 5. Representative pyruvate dehydrogenase assay on cells.
Cells were treated with DCA, MCA and Pro-MCA-NP at 100 μM for 72 h before performing the assay, (A) comparative increase in PDH activity of cells correlated with increase in absorbance of reaction mixture at λ 450 nm where “red circle line” represents PDH activity in untreated cells and (B) time dependent increase in PDH activity of protein (125 μg/ml).
Figure 6
Figure 6. Selective loss of anti-cancer cell growth efficiency of Pro-haloacetate-NPs against non-cancerous cell of breast origin (MCF-10A) compared to cancer cell (MCF-7) of same origin.
Bright field imaging performed on (A) MCF10A cells post 48 h treatment with (B) Lipid-NPs; (C) Pro-DCA-NPs; (D) Pro-MCA-NPs; (E) Pro-DBA-NPs; (F) DCA; (G) MCA and (H) DBA at a concentration of 100 μM revealed no significant loss of cell growth density. (I) A comparative cell growth inhibition study in MCF-7 and MCF-10A cells treated with a concentration of 100 μM of DCA, MCA and DBA in free or form of Pro-haloacetate-NPs supporting biostatistically significant low effective toward MCF-10A cells compared to MCF-7 cells by p < 0.01. Propidium iodide incorporation study on (J) MCF-10A and treated with (K) Pro-DCA-NP; (L) Pro-MCA-NP and (M) Pro-DBA-NP with a haloacetate concentration of 100 μM for 48 h.
Figure 7
Figure 7. In vivo results on xenograft mouse model.
(A) Timeline of experiment; representative animals with tumors after treatment with (B) DPBS; (C) Pro-MCA-NP; (D) Pro-DCA-NP and (E) DCA and (F) representative tumors collected after sacrificing the animals. (G) Tumour growth curves with time and (H) fold change after treatment with Pro-MCA-NP, Pro-DCA-NP and DCA. H&E images of tumour sections for treatments with (I) DPBS; (J) Pro-MCA-NP; (K) Pro-DCA-NP and (L) DCA (Black arrows indicate presence of fragmented nuclei in tumor sections).
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References

    1. Bonnet S. et al.. A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11, 37–51 (2007). - PubMed
    1. Welsh S., Williams R., Kirkpatrick L. & Powis G. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1alpha. Mol. Cancer Ther. 3, 233–244 (2004). - PubMed
    1. Deus C. M., Coelho A. R., Serafim T. L. & Oliveira P. J. _targeting mitochondrial function for the treatment of breast cancer. Fut. Med. Chem. 6, 1499–1513 (2014). - PubMed
    1. Gopinath S. & Evangelos M. D. Pyruvate dehydrogenase kinase as a novel therapeutic _target in oncology. Front. Oncol. 3, 38 (2013). - PMC - PubMed
    1. Ruggieri V. et al.. Dichloroacetate, a selective mitochondria-_targeting drug for oral squamous cell carcinoma: a metabolic perspective of treatment. Onco_target. 6, 1217–1230 (2015). - PMC - PubMed

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