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UPREGULATION OF GLYCOLYSIS IN PANCREATIC CANCER CELLS UNDER HYPOXIA INHIBITS THE ANTITUMOR EFFECT OF ASCORBIC ACID MEDIATED BY REACTIVE OXYGEN SPECIES
Takeru Maekawa
*, Toru Miyake, Ken-ichi Mukaisho, Masaji Tani, Shinji Uemoto
Surgery, Shiga Ika Daigaku, Otsu, Shiga, Japan
Background. Pancreatic cancer (PC) is a highly lethal cancer, so it is essential to develop a novel therapy. Ascorbic acid (AA) is a naturally occurring substance that exhibits antitumor effects but has not yet been used clinically. We investigated the antitumor effect of AA on PC.
Method. BALB/c-nu/nu mice injected with the T3M4, the human pancreatic cancer cell line, were treated with 2 g/kg AA intraperitoneally twice a week. Tumor volume and weight were compared between control (n=6) and AA groups (n=7).
In vitro, the effects of AA on cell viability were measured by Annexin V assay under hypoxia or in the presence of catalase (100 ?g/mL), the antioxidant agent, in the glucose-conditioned medium. The effect of AA on creating reactive oxygen species (ROS) was measured by 2,7-dichlorodihydrofluorescein diacetate assay. To detect the oxidative stress reaction of tumor cells, western blot analysis for p38 MAPK and JNK was performed. RNA-seq was performed in 25 mM glucose medium in four groups: control, ten mM AA, hypoxia (O2: 1 %), and both.
Result. In vivo, AA did not reduce tumor volume (1694 mm
3 vs 1421 mm
3, p=0.3894) and weight (1583 mg vs 1192 mg, p=0.2993).
In vitro, AA induced cell death of T3M4 in 25 mM glucose medium (percentage of Annexin V positive cells: 7.3 % vs 42.0 %, p=0.0019), but not in the presence of catalase. Western bolting showed that both p38 MAPK and JNK, the major of oxidative stress reactions, were activated by AA. However, the antitumor effects of AA were inhibited under hypoxia (Figure 1). ROS detection assay showed that AA created ROS in both normal (fold change in ROS: 8.8, p<0.0001) and hypoxic environments (fold change in ROS: 7.3, p=0.0001), indicating that the antitumor effect of AA was suppressed by a different method than that of suppressing ROS production. Next,
in vitro, AA exhibited antitumor effects under hypoxia in glucose-restricted conditions (1 mM) (Figure 1). RNA-seq showed that the KEGG: TNF signaling pathway was enriched (gene count:6, FDR?0.0001) in the AA group compared to the control group, although it was not enriched in the combination group. RNA-seq also showed that the KEGG: HIF-1 signaling pathway was enriched (gene count:27, FDR?0.0001) in the combination group compared to the control group. Gene set enrichment analysis revealed positive enrichment of glycolysis genes in the combination group, and the volcano plots also showed increased expression of genes important for glycolysis, such as SLC2A1 and SLC2A3 (Figure 2).
Conclusion. Ascorbic acid exhibited antitumor effects via ROS-mediated pathway and was inhibited under hypoxia. Activation of glycolysis under hypoxia played an important role in inhibiting the antitumor effects of AA.
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