Low-dose YC-1 combined with glucose and insulin selectively induces apoptosis in hypoxic gastric carcinoma cells by inhibiting anaerobic glycolysis

Kota Wakiyama,Jun Nakamura,Masao Kaneki,Hirokazu Noshiro,Shinichi Aishima,Yoshihiko Kitajima,Tomokazu Tanaka,Kazuyoshi Yanagihara

doi:10.1038/s41598-017-12929-9

Kota Wakiyama, Jun Nakamura + Show 6 more

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https://doi.org/10.1038/s41598-017-12929-9

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Abstract

This study aimed to establish a therapeutic strategy targeting hypoxic cancer cells in gastric carcinoma (GC). YC-1 is a HIF-1α inhibitor, and we revealed that low-dose YC-1 (10 µM) suppressed HIF-1α expression, and induced hypoxia-dependent apoptosis in the GC cell line 58As9. This hypoxia-specific apoptosis induction by YC-1 involved excessive reactive oxygen species (ROS) generation. The apoptotic effect of 10 µM YC-1 was enhanced by additional glucose (G) and insulin (I) treatments. RT-PCR demonstrated that 10 µM YC-1 reduced hypoxia-induced expression of HIF-1α targets involved in anaerobic glycolysis. Metabolic analysis showed that YC-1 shifted glucose metabolism in hypoxic cells from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). Additional GI accelerated membranous GLUT1 translocation, elevating glucose uptake, and increased acetyl-CoA levels, leading to more ROS generation in hypoxic YC-1-treated cells. Finally, we evaluated the anti-cancer effect of low-dose YC-1 (1 mg/kg) + G (2 g/kg) and I (1 unit/3 g G) treatment in xenograft models. YC-1 + GI therapy strongly inhibited tumour growth. Immunohistochemical analysis demonstrated that YC-1 + GI reduced HIF-1α expression and pimonidazole accumulation in tumours. Conversely, YC-1 + GI increased intra-tumoral 8-OHdG and levels of apoptosis markers. Low-dose YC-1 + GI is a unique therapy targeting hypoxic GC cells that generates lethal ROS via forced activation of OXPHOS.

Highlights

Intratumoral hypoxia is a common characteristic of many solid tumours[1,2]
Fold change (FC) of hypoxia/normoxia is presented at the bottom. (b) Cell death rate in YC-1, GI- and YC-1 + GI-treated cells under normoxia or hypoxia for 3 d. (c) Western blot analysis of cleaved-caspase[3] and cleaved-PARP in YC-1, YC-1 + G, YC-1 + I- and YC-1 + GI-treated cells under hypoxia for 24 h. β-actin was expressed in all cells. ns: not significant, *p < 0.05, **p < 0.01
Western blot (WB) analysis showed that HIF-1α was elevated by hypoxia in control cells, while the hypoxic induction of HIF-1α was inhibited in YC-1-treated 58As9 and KD cells (Fig. 1c)

Summary

Introduction

Intratumoral hypoxia (low O2) is a common characteristic of many solid tumours[1,2]. HIF-α (HIF-1α or HIF-2α), a basic-helix-loop-helix transcription factor, functions as a master regulator of oxygen homeostasis. We reported that HIF-1α knockdown (KD) by siRNA induces apoptosis in the gastric carcinoma (GC) cell line 58As9 under hypoxia This hypoxia-dependent apoptosis was induced by excessive production of reactive oxygen species (ROS), whereby HIF-1α KD inhibited hypoxic induction of genes involved in the ROS www.nature.com/scientificreports/. It has been reported that ROS are increased in hypoxic cancer cells, and HIF-1α induction plays an adaptive mechanism in controlling ROS generation via up-regulating genes involved in anaerobic glycolysis[3,15,16,19]. Thereafter, LDHA alternatively converts pyruvate to lactate and MCT4 effluxes the lactate[24,25,26] Together, these reports indicate that HIF-1α is a central molecule in suppressing excessive ROS production in hypoxic cells via up-regulating target genes involved in anaerobic glycolysis. No study has assessed the anti-cancer effect of YC-1 on cancer metabolism under hypoxia

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