Lapacho tree
Lapacho tree

β-lapachone (β-lap), a novel anti-tumor quinone extracted from the bark of the lapacho tree , has shown great potential as a chemotherapeutic agent for treatment of human breast, pancreatic, prostate and non-small cell lung cancers that overexpress NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase.

NQO1 "bioactivates" β-lap which induces a unique cell death pathway that involves hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme that senses DNA damage. PARP-1 hyperactivation by β-lap is induced via a combination of reactive oxygen species formation and calcium released from the endoplasmic reticulum (ER) of NQO1 overexpressing cells, which results in DNA SSB.

Stained cells
β-lap induces SSBs and causes un-repairable DNA damage. Alkaline comet assays (which detect SSBs and DSBs) and neutral comet assays (which detect only DSBs) were performed on human prostate cancer cells (PC-3) unexposed or exposed to β-lap for the indicated times.

Hyper-activation of PARP-1 causes a dramatic loss of nucleotide (NAD+/ATP) pools, which in turn, results in loss of intracellular calcium homeostasis and subsequent activation of µ-calpain, a pro-apoptotic cysteine protease. β-lap irreversibly kills tumor cells overexpressing NQO1 , while sparing NQO1-deficient cells.

Stained cells
PC-3 cells were exposed to 4 µM β-lap or 20 Gy IR and then alkaline comet assays were performed at various times after exposure to β-lap or IR.

The compound is also a potent radiosensitizer in tumors expressing elevated levels of NQO1. Further investigation has shown that β-lap exposure induces an NQO1-dependent activation of MRE11, Rad50 and Nbs-1.

MCF-7 breast cancer cells exposed to β-lap and stained with antibodies to MRE11, Rad50, Nbs-1-p and g-H2AX.

In addition, ATM serine 1981, DNA-PKcs threonine 2609, and Chk1 serine 345 phosphorylation were noted, indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated β-lap lethality (Bentle et al., Cancer Res., 2007). These studies give new insight into the mechanism by which β-lap radiosensitizes cancer cells and suggests that NHEJ is a potent target for enhancing the therapeutic efficacy of β-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels.

Our laboratory has devoted decades to investigating the underlying killing mechanism of ß-lap and to improve the efficacy of this drug for clinical applications. Working with the laboratory of Dr. Jinming Gao, we have been able to increase the solubility, bioavailability and efficacy of ß-lap using nanoparticle delivery vehicles. Delivery of targeted β-lap encapsulated nanoparticles and the combination of these with IR are at the forefront of our current investigations for the treatment of cancers overexpressing NQO1.

Imaging Experiments

MRI image
MRI splice of a mouse with pancreatic tumors showing detection of a [1-13C]lactate signal in the tumors after injection of hyperpolarized [1-13C]pyruvate.

Hyperpolarization experiments allows for non-invasive imaging of metabolism in tumor cells. The conversion of [1-13C]pyruvate to [1-13C]lactate can be assessed using techniques such as NMR for in vitro experiments and MRI imaging for in vivo models. The [1-13C]lactate signal detected after a [1-13C]pyruvate injection can be used to measure the metabolic flux in cancer cells and will allow for determination of b-lapachone’s effect on tumor metabolism in NQO1 +/- models.