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Squalene deters drivers of RCC disease progression beyond VHL status

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Abstract

Identifying drug candidates to target cellular events/signaling that evades von Hippel-Lindau tumor suppressor (VHL) gene interaction is critical for the cure of renal cell carcinoma (RCC). Recently, we characterized a triterpene-squalene derived from marine brown alga. Herein, we investigated the potential of squalene in targeting HIF-signaling and other drivers of RCC progression. Squalene inhibited cell proliferation, induced cell dealth and reverted the cells' metastatic state (migration, clonal expansion) independent of their VHL status. Near-identical inhibition of HIF-1α and HIF-2α and the regulation of downstream targets in VHL wild type and mutant cell lines demonstrated squalene efficacy beyond VHL-HIF interaction. In a rat model of chemically induced RCC, squalene displayed chemopreventive capabilities by substantial reversal of lipid peroxidation, mitochondrial redox regulation, maintaining ∆ψm, inflammation [Akt, nuclear factor κB (NF-κB)], angiogenesis (VEGFα), metastasis [matrix metalloproteinase 2 (MMP-2)], and survival (Bax/Bcl2, cytochrome-c, Casp3). Squalene restored glutathione, glutathione reductase, glutathione-s-transferase, catalase, and superoxide dismutase and stabilized alkaline phosphatase, alkaline transaminase, and aspartate transaminase. The correlation of thiobarbituric acid reactive substance with VEGF/NF-κB and negative association of GSH with Casp3 show that squalene employs reduction in ROS regulation. Cytokinesis-block micronuclei (CBMN) assay in VHLwt/mut cells revealed both direct and bystander effects of squalene with increased micronucleus (MN) frequency. Clastogenicity analysis of rat bone marrow cells demonstrated an anti-clastogenic effect of squalene, with increased polychromatic erythrocytes (PCEs), decreased MNPCE,s and MN normochromatic erythrocytes. Squalene could effectively target HIF signaling that orchestrate RCC evolution. The efficacy of squalene is similar in VHLwt and VHLmut RCC cells, and hence, squalene could serve as a promising drug candidate for an RCC cure beyond VHL status and VHL-HIF interaction dependency. Summary: Squalene derived from marine brown algae displays strong anti-cancer (RCC) activity, functionally targeting HIF-signaling pathway, and affects various cellular process. The significance of squalene effect for RCC is highlighted by its efficiency beyond VHL status, designating itself a promising drug candidate.

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Data availability

All data pertaining to this study is presented within this manuscript and in the supplementary figures.

Abbreviations

ALP:

alkaline phosphatase

ALT:

alkaline transaminase

AST:

aspartate transaminase

BLM:

bleomycin

BSE:

bystander effect

BUN:

blood urea nitrogen

Casp3:

caspase-3

CAT:

catalase

CBMN:

cytokinesis-block micronuclei

ccRCC:

clear cell RCC

CDNB:

1,chloro-2,4,dinitrobenzene

DAPI:

4′,6-diamidino-2-phenylindole

DE:

direct effect

DTNB:

dithionitrobenzene

EA:

ethylacetate

ELISA:

enzyme-linked immunosorbent assay

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GR:

glutathione reductase

GSH:

glutathione

GST:

glutathione-s-transferase

H&E:

hematoxylin and Eosin

HIF:

hypoxia inducible factor

HIF1α:

hypoxia inducible factor 1 α alpha

HIF2 α:

hypoxia inducible factor 2 α

ICI:

immune checkpoint inhibitor

IHC:

immunohistochemistry

KBrO3 :

potassium bromate

KCl:

potassium chloride

LPO:

lipid peroxidation

MAPK:

mitogen-activated protein kinases

MDA:

malondialdehyde

MMP-2:

matrix metalloproteinase 2

MN:

micronuclei

MNNCE:

micronuclei normochromatic erythrocytes

MNPCE:

micronuclei polychromatic erythrocytes

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NF-κB:

nuclear factor kappa B

pAkt:

phospho-specific AKT at Ser473

PCE:

polychromatic erythrocytes

PMS:

post-mitochondrial supernatant

qPCR:

quantitative polymerase chain reaction

RCC:

renal cell carcinoma

ROS:

reactive oxygen species

SOD:

superoxide dismutase

SQ:

squalene

SRB:

sulforhodamine B

TBA:

thiobarbituric acid

TBARS:

thiobarbituric acid reactive substance

TCA:

trichloroacetic acid

TNFα:

tumor necrosis factor α

VEGFα:

vascular endothelial growth factor α

VHL:

von Hippel-Lindau

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Acknowledgments

The authors thank Central Research Facility, Sri Ramachandra Institute of Higher Education and Research; Dr. Arulkumaran Shanmugam, Microbiology and Immunology, New York Medical College; and Dr. T. Radhiga and Dr. V.C. Renju, Department of Medical Biochemistry, Annamalai University for their support with laboratory access, reagents, and antibodies. The authors also acknowledge the OUHSC Staff Editor (Ms. Kathy Kyler) for the help in critically reviewing this manuscript.

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Funding

Supported by the Department of Science and Technology (DST No: SR/SO/AS-04/2005) and the RMMCH clinical trial projects, Annamalai University, India, for the financial support (project assistant) to Dr. Karthikeyan Rajamani, and Department of Biotechnology, Ministry of Science and Technology (BT/PR11535/AAQ/03/431) to Dr. Somasundaram S Thirugnanasambandan.

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Authors and Affiliations

  1. Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, 608502, TN, India

    Karthikeyan Rajamani, Somasundaram S. Thirugnanasambandan & Balasubramanian Thangavel

  2. Rajah Muthiah Medical College, Annamalai University, Annamalai Nagar, Chidambaram, 608002, TN, India

    Karthikeyan Rajamani, Chidambaram Natesan & Sethupathy Subramaniam

  3. WHO Collaborating Center for Occupational and Environmental Health, ICMR Center for Air Quality, Climate and Health, Department of Environmental Health Engineering, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, TN, 600116, India

    Karthikeyan Rajamani

  4. Department of Radiation Oncology, University of Oklahoma Health Sciences Center, BMSB 737, 947 Stanton L. Young Boulevard, Oklahoma City, OK, 73104, USA

    Natarajan Aravindan

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  1. Karthikeyan Rajamani
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  2. Somasundaram S. Thirugnanasambandan
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  3. Chidambaram Natesan
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  5. Balasubramanian Thangavel
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  6. Natarajan Aravindan
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Contributions

Designed the experiments: KR, ST, and NA. Shared their labs/reagents/kits: CN, SS, BT, and NA. Performed the experiment: KR and ST. Analyzed data: KR, ST, and NA. Manuscript draft: KR, NA, ST, CN, BT, and SS.

Corresponding authors

Correspondence to Karthikeyan Rajamani or Natarajan Aravindan.

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Competing interests

The authors declare that they have no competing interests.

Ethics approval

All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No.80-23 revised 1996) and approved (160/PO/ReBi/S/99/CPCSEA) by the Rajah Muthiah Institute of Medical Science, Institutional Animal Ethical Committee (IAEC)/Institutional Review Board (IRB), Annamalai University, Medical College, Annamalai Nagar.

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Highlights

• Seaweed-derived squalene inhibits RCC cell survival and prompts DNA fragmentation and apoptosis.

• Squalene targets HIFs expression and HIF targets independent of the VHL status of the RCC cells.

• Squalene regulates mitochondrial ROS and lipid peroxidation and targets inflammatory signaling while prompting apoptotic machinery.

• Squalene exerted anti-genotoxic and anti-clastogenic effects in RCC cells and, together, could serve as a promising drug candidate for RCC treatment.

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Rajamani, K., Thirugnanasambandan, S.S., Natesan, C. et al. Squalene deters drivers of RCC disease progression beyond VHL status. Cell Biol Toxicol 37, 611–631 (2021). https://doi.org/10.1007/s10565-020-09566-w

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  • DOI: https://doi.org/10.1007/s10565-020-09566-w

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