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Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling

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Abstract

It has been well established that radiation-induced gastrointestinal injury is manifested through loss of intestinal crypt stem cells and disruption of the mucosal layers, resulting in diarrhoea, weight loss, electrolyte imbalance, infection and mortality. Podophyllotoxin and rutin in combination (G-003M) has been reported to regulate endogenous cellular antioxidant defense systems and inflammatory response. However, the mechanism by which G-003M ameliorates radiation-induced intestinal stem cell (ISC) injury remains unclear. Here, we hypothesize the radioprotective potential of G-003M would amplify the intestinal crypt stem cells through upregulation of Wnt/β-catenin signaling and accelerate the reconstitution of the irradiated intestine. Our results showed significant functional and structural intestine regeneration in irradiated animals following G-003M treatment which resulted in improved animal survival. Immunohistochemical examination revealed an enhancement in Lgr5+ ve crypt stem cells. Increased β-catenin nuclear translocation resulted in upregulation of β-catenin target genes that supported ISC renewal and expansion in G-003M-treated mice, as compared to IR-treated mice. However, G-003M could not rescue the Wnt knockdown cohorts (XAV939 treated) which exhibited greater incidence of intestinal apoptosis, DNA damage and crypt depopulation upon radiation exposure. These findings suggest the involvement of Wnt pathway during G-003M mediated amelioration of IR-induced ISC injury. G-003M also minimised acute inflammation by restricting the infiltration of immune cells into the intestinal venules. Furthermore, G-003M treated animals showed improved anti-tumor response compared to FDA approved Amifostine. Taken together, our findings suggest that G-003M may be used as a potential countermeasure for radiation injuries as well as an adjuvant during anti-cancer therapy.

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Acknowledgements

We extend our sincere gratitude to Dr. Ajay Kumar Singh, Director, INMAS for providing necessary infrastructure and support to accomplish this work. We also thank Dr. B.G. Roy for providing experimental animals for the study. The support from Ms Anjali Sharma and Ms Namita Kalra for irradiation facility and flow cytometry measurements is duly acknowledged. BK, RR acknowledges UGC for the award of senior research fellowship.

Funding

This work was funded by the grant INM-313 from Defence Research & Development Organization (DRDO), Ministry of Defence, Govt. of India. BK, RR acknowledges University Grants Commission (UGC), Governtment of India for the award of senior research fellowship. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. Division of Radioprotective Drug Development and Research, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Majumdar Marg, Timarpur, Delhi, 110054, India

    Bhargab Kalita, Rajiv Ranjan & Manju Lata Gupta

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  1. Bhargab Kalita
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BK MLG: Conceived, designed and performed the experiments. RR BK: Analyzed the data. RR BK: Contributed reagents/materials/analysis tool. MLG BK: Wrote the paper.

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Correspondence to Manju Lata Gupta.

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Kalita, B., Ranjan, R. & Gupta, M.L. Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling. Apoptosis 24, 326–340 (2019). https://doi.org/10.1007/s10495-019-01519-x

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