During tumorigenesis, the highly metabolic tumor cells require a steady supply of O2
and nutrients to thrive and proliferate, which is usually associated with angiogenesis and regulatory growth factors. During later stages of tumor growth, a discrepancy of O2
metabolism leads to concentrated areas with lower O2
partial pressures and a disorderly microvasculature, which further produces a microenvironment depleted of O2
and blood flow.23–25
Under these harsh conditions, tumor cells experience a switch in their metabolic machinery to survive by relying on glucose consumption and anaerobic glycolysis, which is a less efficient method of ATP production than oxidative phosphorylation.26
To conserve energy, the hypoxic cells proliferate at a much slower rate than the typical abnormal tumor cells; therefore, becoming resistant to antiangiogenic, chemotherapeutic, and radiation therapy treatments that target the rapidly dividing cells.27
Because these cells become heavily dependent on glucose consumption under anaerobic conditions, therapeutic agents that obstruct glycolysis have been explored. It has been shown that the glycolytic inhibitor 2-DG effectively targets these hypoxic cells in retinoblastoma tumors in vivo5,10
and in osteosarcoma (143B) and knockout mtDNA mutant cells in vitro.9
Moreover, a combination therapy of 2-DG and chemotherapy (i.e., carboplatin), significantly reduced tumor burden in LHBETA
retinal tumors more efficiently than when either treatment was provided alone.5
Because the commercially available 2-FG was demonstrated to be a more effective glycolytic inhibitor in vitro than 2-DG in targeting hypoxic cells,9
in the present study we tested the efficacy of focal, periocular delivery of 2-FG in vivo in the LHBETA
transgenic mouse model of retinoblastoma.
In the present study, focal delivery of 2-FG decreased hypoxia after 2-FG injections with minimal toxicities associated with the treatment. Results suggest that longer, biweekly treatments (i.e., 2-FG treatment for 3 weeks) in later stages of tumor growth produce a significant reduction on tumor hypoxia and tumor burden at the dosages used. This outcome demonstrates that 2-FG is a potent glycolytic inhibitor in targeting hypoxic cells in retinoblastoma and further corroborates our previous results showing that glycolytic inhibitors can be used as adjuvant to standard treatment modalities (e.g., chemotherapy) to target the chemoresistant, hypoxic cells. Moreover, histopathological examinations of H&E staining of the LHBETATAG retinal tumor sections did not display any toxicity or abnormality in the eye associated with drug delivery, further revealing that periocular delivery of this drug is a viable and efficient treatment modality.
Furthermore, we analyzed the effects of 2-FG in the different areas of the tumor. As previously characterized, the basal and central regions of retinal tumors display the highest amount of hypoxia, whereas the other regions (i.e., apex, lateral) show little to no hypoxia.5
This study is the first to show that 2-FG decreases the amount of intratumoral hypoxia in vivo in all the different areas analyzed. Our results showing that the basal regions had the greatest percentage of reduced hypoxia may be explained by the findings that control eyes present with a much higher density of hypoxic cells in these areas. Further, basal regions are potentially exposed to higher drug concentrations.
A proposed mechanism of 2-FG in effectively eliminating hypoxic cells is that it interferes with glycolysis through competitively inhibiting phosphoglucose isomerase (PGI) and allosterically inhibiting hexokinase. It is hypothesized that because tumor cells have to sustain high demands of ATP necessary for their development, they express higher levels of the hexokinase but not PGI.28,29
Glycolysis inhibition seems to be a promising tool to target the hypoxic cells that are associated with chemotherapy and radiation therapy resistivity, by interfering with the glycolytic pathway through inhibition of these enzymes.5
Data indicate that the competitive inhibiton of PGI occurs at a lower Km
for 2-DG and perhaps 2-FG than that of hexokinase. Thus, there appears to be a hierarchy where either of these glycolytic inhibitors primarily affect PGI and only at significantly higher concentrations affect hexokinase. If indeed hexokinase is shut down then glucose-6-phosphate levels required for the functioning of the pentose phosphate shunt will be too low. If the pentose shunt is blocked effects on fatty acid synthesis and glutathione function would be expected to be detrimental to the cell.
In addition, it has also been reported that angiogenesis, the development of new blood vessels from pre-existing ones, is also affected with the glycolytic inhibitor 2-DG in the LHBETA
transgenic mouse model.30
We have previously reported that the mammalian target of rapamycin (mTOR), which is an upstream regulator of glycolysis, targeted mature blood vessels in the same animal model.31
These facts suggest that drugs implicated in glucose catabolism have an effect on angiogenesis and/or blood vessel maturation. In the present study, treatment with 2-FG effectively decreased both the percentage of new, mature, and total blood vessels in the retinal tumors of the transgenic animal model of retinoblastoma. Previous reports in which 2-DG showed anti-angiogenic activity in vitro in HUVEC cells was revealed to be due to 2-DG's activity as a mannose mimetic interfering with oligosaccharide synthesis resulting in endoplasmic reticulum stress mediated apoptosis. Thus, it appeared that 2-DG's dual activity as inhibitor of glycosylation as well as glycolysis contributed to its overall activity in reducing hypoxic tumor cells. 2-DG's glycolytic inhibitor analog 2-FG was previously shown to be more potent in inhibiting glycolysis than 2-DG and thereby killing hypoxic cells in vitro.9
Because in the present study 2-FG targets both new and mature blood vessels, it is possible that 2-FG has a dual action on glycolysis and growth factors associated with blood vessel development, thus, affecting several mechanisms associated with tumor cell development under hypoxic conditions in vitro.
The present results substantiate previous in vitro results that the glycolytic inhibitor 2-FG selectively targets the chemoresistant, hypoxic cells in LHBETA
retinal tumors. Because the effects of 2-FG in the present study mimic those of 2-DG, these results also validate the use of glycolytic inhibitors in the treatment of retinoblastoma. The study additionally provides the rationale for using the retinoblastoma model in the future evaluations of glycolytic inhibitors in combination with other drugs (i.e., chemotherapy and radiation therapy) in several cancers.9
In conclusion, our results further demonstrate that hypoxic regions are most pronounced during later stages of the disease that develop in the LHBETATAG transgenic mice containing retinal tumors. This is the first study to elucidate that periocular administration of 2-FG in this animal model produces an impact on hypoxia and a concomitant influence on tumor burden control. This study correspondingly validates our previous trials revealing that glycolytic inhibitors favor the targeting of the chemoresistant, hypoxic cells in retinoblastoma tumors. The future application of periocular 2-FG as an adjuvant treatment to standard chemotherapeutic agents can be explored to potentially enhance the treatment of pediatric retinoblastoma.