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Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 392, Issue 10, pp 1239–1256 | Cite as

PHD-2 activation: a novel strategy to control HIF-1α and mitochondrial stress to modulate mammary gland pathophysiology in ER+ subtype

  • Uma Devi
  • Manjari Singh
  • Subhadeep Roy
  • Avinash C. Tripathi
  • Pushpraj S. Gupta
  • Shailendra K. Saraf
  • Md. Nazam Ansari
  • Abdulaziz S. Saeedan
  • Gaurav KaithwasEmail author
Original Article

Abstract

Estrogen receptor–positive mammary gland carcinoma and its involvement in regulation of overexpressed hypoxia-inducible factor-1α and fatty acid synthase level in hypoxia influenced cancer cells are the present molecular crosstalk of this entire study. To test the hypothesis, we have proceed our study through chemical activation of prolyl hydroxylase 2 which leads to inhibition of hypoxia-inducible factor-1α and fatty acid synthase in ER+MCF-7 cancer cell line and n-methyl-n-nitrosourea induced mammary gland carcinoma rat model. ER+MCF-7 cells were evident with array of nuclear changes when stained through acridine orange/ethidium bromide. Afterward, JC-1 staining of the cells was evident in mitochondrial depolarization. The cells were arrested in G2/M phase when analyzed with flow cytometry. The morphological analysis of rat mammary gland tissue revealed decrease in alveolar buds, restoration of histopathological features along with intra-arterial cushion. The western blotting and fold change expressions of the genes validating the anticancer efficacy of BBAPH-1 is mediated through mitochondria-mediated apoptosis pathway. BBAPH-1 also modulates the expression of prolyl hydroxylase-2 with significant curtailment of hypoxia-inducible factor-1α, fatty acid synthase expression, and their respective downstream markers. These finding suggest that the BBAP-1-mediated activation of prolyl hydroxylase-2 significantly decreased the level of hypoxia-inducible factor-1α and fatty acid synthase. BBAPH-1 also activates the mitochondria-mediated death apoptosis pathway.

Keywords

2-Oxoglutarate N-methyl-n-nitrosourea Prolyl hydroxylase-2 Breast cancer Hypoxia 

Abbreviations

AO

Acridine orange

AB

Alveolar buds

BSA

Bovine serum albumin

CEC

Cuboidal epithelial cells

DTT

Dithiothreitol

DMSO

Dimethyl sulphoxide

DCT

Dense connective tissue

DF

Differentiation score

EtBr

Ethidium bromide

ECG

Electrocardiogram

EBSS

Eagle’s balanced salt solution

FASN

Fatty acid synthase

FBS

Fetal bovine serum

FACS

Fluorescence-activated cell sorter

GSH

Glutathione

HIF

Hypoxia-inducible factor

HRV

Heart rate variability

HR

Heart rate

H&E

Hematoxylin and eosin

HBSS

Hank’s balanced salt solution

HF

High frequency

LF

Low frequency

LCT

Loose connective tissue

MNU

n-Methyl-n-nitrosourea

MC

Myoepithelial cells

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

OPD

o-Phenylenediamine

2-OG

2-Oxoglutarate

PHD-2

Prolyl hydroxylase-2

PI

Propidium iodide

PC

Protein carbonyl

PBS

Phosphate buffer saline

ROS

Reactive oxygen species

SEM

Scanning electron microscope

SOD

Superoxide dismutase

TEST

Toxicity estimation software tool

TRU

Turbidity reduction unit

TBARs

Thiobarbituric acid reactive substances

VEGF

Vascular endothelial growth factor

pVHL

von Hippel–Lindau

Notes

Authors’ contributions

UD and MS carried out the bench work; SR performed the immunoblotting assay and cell culture; ACT performed the in silico studies; PSG evaluated all the data; SKS evaluated the in silico studies; MNA and ASS performed the statistical analysis of the data; GK supervised, designed, and proofread the final manuscript.

Funding information

MS is supported by the University Grants Commission, Government of India, through senior research fellowship (RGNF-2013-14-SC-UTT-38150). SR is supported by the Department of Science and Technology, Government of India, through senior research fellowship (SB/EMEQ-254/2013).

Compliance with ethical standards

The animal experimental procedures were approved by the Institutional Animal Ethics Committee (UIP/IAEC/2014/FEB/16), United Institute of Pharmacy, Allahabad, and performed as per the guidelines laid by Department of Animal Welfare, Government of India. Tumor

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

210_2019_1658_MOESM1_ESM.jpg (304 kb)
Fig. S1: Docking study and toxicity profile of BBAPH-1. BBAPH-1 was docked with PHD-2 protein (PDB id-2G19). The binding energy was found −3.97 kcal/mol. The hydrogen bond was observed between UNK 0: H1 and ARG 322: HH12 1. The amino acids attached to protein were LEU 343, VAL 376, TYR 303, HIS 374, TYR 310, MET 299, TRP 389, ASP 315, ARG 322.Toxicity profile of BBAPH-1 was estimated with TEST which revealed that BBAPH-1 was found to be non-mutagenic. (JPG 304 kb)
210_2019_1658_MOESM2_ESM.jpg (26 kb)
Fig. S2: Metabolic profiling of BBAPH-1. The metabolic profiling of BBAPH-1 shows CYP450 mediated hydroxylation at 6th, 10th, 19th and 20th carbon position. (JPG 26 kb)
210_2019_1658_MOESM3_ESM.jpg (494 kb)
Fig. S3: Representative ECG tracing of the animals treated with MNU and BBAPH-1. I- Control (0.9% normal saline, p.o.); II- Toxic control (MNU, 8 mg/kg, i.v.); III-BBAPH-1+ MNU (56.62 μg/kg, s.c + 8 mg/kg MNU, i.v.); IV- BBAPH-1+ MNU (113.25 μg/kg, s.c + 8 mg/kg MNU, i.v.). (JPG 494 kb)
210_2019_1658_MOESM4_ESM.docx (12 kb)
Table S1 (DOCX 11 kb)
210_2019_1658_MOESM5_ESM.docx (11 kb)
Table S2 (DOCX 11 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Uma Devi
    • 1
  • Manjari Singh
    • 2
  • Subhadeep Roy
    • 2
  • Avinash C. Tripathi
    • 3
  • Pushpraj S. Gupta
    • 1
  • Shailendra K. Saraf
    • 3
  • Md. Nazam Ansari
    • 4
  • Abdulaziz S. Saeedan
    • 4
  • Gaurav Kaithwas
    • 2
    Email author
  1. 1.Department of Pharmaceutical Sciences, Faculty of Health and Medical SciencesSam Higginbottom Institute of Agricultural Sciences and TechnologyAllahabadIndia
  2. 2.Department of Pharmaceutical SciencesBabasaheb Bhimrao Ambedkar University (A Central University)LucknowIndia
  3. 3.Faculty of Pharmacy, Babu Banarsi Das Northern India Institute of TechnologyBabu Banarsi Das UniversityLucknowIndia
  4. 4.Department of Pharmacology, College of PharmacyPrince Sattam Bin Abdulaziz UniversityAl-KharjSaudi Arabia

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